Nal de Patentes Areas Mecanica, Elos de Utilidad Area Electrica MX/a/2017/008919

NAL DE PATENTES
AREAS MECANICA,
ELOS DE UTILIDAD
AREA ELECTRICA
MX/a/2017/008919
5 de febrero de 2019.
ey de la Propiedad
iguiente:
fueron presentadas
equisitos No. 72923
11 de diciembre de
ntados en su escrito
No. 72923 del 8 de
17, contiene materia

de la invención, ya
ANTENTE BREVE
, , ¡cas que no estaban
presentes en la solicitud de patente MX/a/2017/008919 como originalmente se presentaron ante el IMPI. Dichas
características no se encuentran contenida en la materia originalmente presentada en su conjunto de su solicitud de
MX/2019/8369
_
__
r en qué parte de Ia
onan La figura 1 es
escala de grises y
60 años, causa de
ar los resultados del
que existe entre un
es una imagen que
dos filtros descritos
de muerte; cancer
s decir que tiene un
e igual intensidad a
ura 3 es una imagen
determinados filtros
80 años, causa de
un rango desde los
pendiendo la luz de
La figura 4 es una
manos de indiviudo
laridad u oscuridad,
as en las figuras 1,
defecto
o bien, eliminar la
ISUALES PRUEBA
antes mencionado.
n fundamento en el
las Reglas para la
on con fundamento
. , . 'te una excitación de

longitud de onda de 372 nm y una emisión de 456 nm dentro del denominado U que refiere a Ultravioletas; un filtro
o cubo de excitación ; sin embargo, el término o es indefinido y como tal no deja claro el alcance de dicha
reivindicación.
3.2 La reivindicación dependiente 5, reclama: "preferentemente se documenta conectando una cámara fotográfica o
de video, al microscopio. ; sin embargo, el término o es indefinido y como tal no deja claro el alcance de dicha
MX/2019/8369 Pág- 2
o, el término o es
determinar si una
a en la fecha de la
que al determinar el
uientes documentos
HMAN DE JUNIO
posición pública en
undial denominada
en el artículo 16 de
3, primer párrafo),
, 4);
a 6 a la página 8 a
6, último párrafo en
onda del 490nm al
ión que permite una
do U que refiere a
Ultravioletas (página 4, figura 2; primer párrafo página 6, último párrafo a la página 8 a primer párrafo); un filtro o
(ver objeción 3.1) cubo de excitación de banda angosta denominado U-MNB SP 470nm-490 nm, DM 500, BA 515
(figura 28; tabla 3).
MX/2019/8369 Pág. 3
D2 si lo hace en el
estado de la técnica
¡dad inventiva
raciones necesarias
ndo las diferencias
ara cumplir así con
rtículo 16 de Ia LPI
aciones anteriores,
(ver objeción 3.3, a
raciones necesarias
8 diferencias entre
cumplir así con lo
reivindicaciones, no
en la solicitud y/o
lo establecido en el
ondiente.
siguiente a la fecha
| plazo señalado se
y 58 de la Ley de la
El presente oficio corresponde al primer requerimiento de examen de fondo, el cual se emite, además de lo
fundamentado anteriormente, conforme al artículo 13 del Acuerdo por el que se establecen Reglas y Criterios para la
resolución de diversos trámites ante el Instituto Mexicano de la Propiedad industrial, publicado en el Diario Oficial de la
Federación el día 9 de agosto de 2004.
MX/2019/8369 Pág- 4
is 2 de la Ley de la
/1994, 25/10/1996,
/2010, 28/06/2010,
'º fracciones I, II, III,
ado el 01/07/2002,
segundo guión, 16
:d Industrial (D.O.F.
rdinador, Directores
: rtamentales y otros
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MX/2019/8369 Pág. 5
DESCRIPTION CN106080004
Abstract The invention discloses a three-dimensional stereoscopic imaging process for human ashes,
comprising the following steps: image acquisition: processing a human body by using three-dimensional
imaging technology to obtain a three-dimensional image; image processing: synthesizing and repairing the
three-dimensional image through a graphic workstation Available 3D data format, through the slicing program,
the discretized model data is obtained and input into the 3D printer; material preparation: the human ashes are
processed into ultrafine powder by the pulverization ultra-fine process, and the diameter of the ultrafine powder
is 350 3000 Imaging treatment: The ultrafine powder is heated and melted, and then sent into a 3D printer to
form a solid portrait of a solid surface through a 3D printer. The ashes statue made by the invention changes
the original funeral customs, not only saves natural resources and social resou rces, but also alleviates the
economic burden of the existing funeral customs.
Three-dimensional stereoscopic imaging process for human ashes
Technical field
The invention relates to a human body ashes imaging method, in particularto a three-dimensional three-
dimensional imaging method for human body ashes, belonging to the ñe|d of funeral technology.
Background technique
According to the applicant's understanding, the funeral methods in most areas of China are cremation. The
implementation of this policy is not only a manifestation of human progress, but also a major measure for the
benefit of the country and the people. However, the current methods of ashes treatment are nothing more than
the following: First, the installation of burial, this burial method is still prevalent in many places in China and
abroad, and the descendents have left the ashes for their safety. Customs of burial; second, the construction of
cemeteries, the use of barren ridges and even the occupation of cemetery to build cemeteries, provinces,
cities, counties and even conditional towns have a large number of operational or public welfare cemeteries;
third, other ways , ashes wall, ash hall, tree burial, flower burial, Iawn burial, river burial and even sea burial,
etc., but no matter what kind of burial method is used, the result still occupies a large amount of available land
and reduces forest coverage. Still mining non-renewable stone mines; still more or less polluted rivers and
oceans.
Summary of the invention
In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to propose a
three-dimensional imaging process for human ashes, which can realize the commemoration of the loved ones,
moum the grief, and avoid occupyíng land and other resources, and also solve the environmental protection.
problem.
The object of the present invention is achieved by the following technical solutions: a three-dimensional
stereoscopic imaging process for human body ashes, comprising the following steps:
I. Image acquisition: image acquisition: using three-dimensional imaging technology to process human external
data acquisition to obtain three-dimensional images;
Il. Image processing: synthesizing and repairing the three-dimensional image through a graphic workstation,
and deriving the available three-dimensional data format, and obtaining the discretized model data through the
slicing program, and inputting the 3D printer;
Ill. Preparation of materials: The human ashes are processed into ultrafine powder by a pulverization and
ultrafine process, and the fineness of the ultrafine powder is 350 to 3000 meshes;
IV. Imaging treatment: After the ultrafine powder is heated and melted, it is sent into a 3D printer to form & solid
portrait with a smooth surface through a 3D printer.
Further, in the foregoing three-dimensional stereoscopic imaging process of the human ashes, the image
acquisition method in the step I includes: a camera matrix system forfinely collecting the appearance features;
3D scanner system for collecting whole body features and applications for scenes where fineness is not
critical;
The three-dimensional stereoscopic image system, through computer aided three-dimensional imaging, is
used for image collection of animals and articles that are preferred by the deceased or their lifetime when the
camera matrix system is inconvenient to use.
Further, in the foregoing human bodyashes three-dimensional imaging process, in step Il, the graphics
workstation is a central computer having a three-dimensional data generation and post processing system.
Further, in the foregoing three-dimensional three-dimensional imaging process of human ashes, in step III, the
human ashes are processed into ultrafine powder by pulverization and ultra-fine processing, and mixed with
non-metal materials to form a mixed process material, and the mixed process materials are Material
consumables are processed into 3D printer consumables. The non-metallic materials are fluorite, plastic,
Iimestone or ore powder with a fineness of 350 to 3000 mesh.
Further, the foregoing three-dimensional three-dimensional imaging process of human ashes, in step IV, is
divided into Iow-melting printing and high-melting printing according to the melting point of the material input
into the 3D printer. Low melting point printing, the heating system is a printing system of an electric heating
melter, and the heating melting temperature is 150 º C - 250 º C. High melting point printing, the heating
system is a printing system of a laser melter, and the heating and melting temperature ¡$ 1400 º C - 2000 º C.
Further, in the foregoing three-dimensional three-dimensional imaging process of human ashes, in step IV, a
color adhesive is added for fuII-color printing.
The outstanding effect of the invention is that the ashes statues produced by the invention can be arranged
into a family hall for future generations to realize, to commemorate the loved ones, to mourn the mouming, and
to change the original funeral customs, not only saving. Natural resources and social resources can also
alleviate the economic burden of existing funeral practices.
The specific embodiments of the present invention will be further described in detail below with reference to the
accompanyíng drawings, so that the technical solutions of the present invention can be more easin understood
and understood.
DRAWINGS
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic flow chart of the present invention.
Detailed ways
Example 1
First, the image acquisition processing of the human body: the camera matrix system is selected. The camera
matrix system includes a camera, a bracket, a flash unit, and a communication cable, and the face is finer
collected by arranging a plurality of digital cameras mounted on the automatic indexing pan/tilt head. feature.
Combined with the 3D scanner system, it collects aII-body features and scenes that require less precision. For
the image acquisition of the camera matrix system, or for the animals and articles that are loved by the
deceased, the three-dimensional stereoscopic image system is used to obtain the three-dimensional original
image through computer aided three-dimensional imaging.
Secondly, the original image data collected in the previous period is synthesized and repaired by the central
computer graphics workstation, or the collected data is secondarin created by the drawing software to match
the real human body, and then the available three-dimensional data format is exported, and the sliced program
is obtained. After the discretized model data is obtained and converted into a format recognizable by the 3D
printer, the 3D printer is input.
The human ashes are placed in a micro-pulverizerfor ultra-fine powder processing, and the ultra-fine powder
particles after completion are 350 mesh to 3000 mesh. Ultrafine powder processing can change the material
properties of the ashes, making them more fluid in high temperature melting. The finer the ashes are
pulverized, the higher the surface finish after imaging. The ultrafine powder particles are heated by a laser
melter to 1400 º C to 2000 º C for melting, and a monochrome DDM printer is selected for printing bye 3D
printer. If you choose fuII-color printing, you will end up with a solid, solid portrait that is the same color as the
3D image.
Example 2
matrix system includes a camera, a bracket, a flash unit, and a communication cable, and the face is finely
The human ashes and non-metal materials are placed in a micro-grinder in a certain proportion and processed
in ultrafine powder. The ratio is determined according to the size after molding, and the ultrafine powder
particles after completion are 350 mesh to 3000 mesh. The mixture is mixed by a mixer, and the ultrafine
powder particles are heated by a laser melter to 1400 º C to 2000 º C for melting, and a monochrome DDM
printer is selected for printing by a 3D printer. The result is a solid portrait with a smooth surface and the same
color as the 3D image.
Example 3
The human ashes and non-metal materials are placed in a micro-grinder in a certain proportion to carry out
ultra-fine powder processing, and the ultra-fine powder particles after completion are 350 mesh to 3000 mesh.
It is mixed by a mixer and processed into a 3D printer consumable using a new material consumable
processor.
The ultrafine powder particles are heated by a laser melter to 1400 º C - 2000 º C for melting, input into a 3D
printer, and a monochrome FDM printer is selected for printing according to requirements, and finally & solid
portrait with a smooth surface and three-dimensional imaging is prepared. Same color.
Example 4
The human ashes are placed in a micro-pulverizerfor ultra-fine powder processing, and the ultra-fine powder
particles after completion are 350 mesh to 3000 mesh.
The ultra-fine powder particles are made into consumables for use in 3D printers and placed in a 3D printer. At
the same time, a color adhesive is added to the 3D printer, and a fuII-color 3DP printer is selected for fuII-color
printing according to the requirements, and finally a solid portrait with a smooth surface is formed, which is the
same color as the three-dimensional image.
Example 5
The human ashes and the non-metallic materials are placed together in a micro-pulverizer for ultra-fine powder
processing, and the mixing amount of human ashes and non-metal materials is selected according to the size
after molding, and the ultrafine powder particles after completion are 350 mesh to 3000. Head. It is mixed by a
mixer and processed into a 3D printer consumable by a material consumable processor.
Make ultra-fine powder particles into consumables for 3D printers, put them into 3D printers, and add color
adhesives to 3D printers. SelectfuII-color 3DP printers for fuII-color printing according to requirements, and
finally make the surface smooth. The physical portrait is the same color as the 3D image.
Example 6
The human ashes and the non-metallic materials are placed together in a micro-pulverizer for ultra-fine powder
processing, and the mixing amount of human ashes and non-metal materials is selected according to the size
after molding, and the ultrafine powder particles after completion are 350 mesh to 3000. Head. It is mixed by a
mixer and processed into a 3D printer consumable by a material consumable processor.
The ultrafine powder particles are heated to 150 º C - 250 º C by electric heating and melting the melter, and
the consumables that can be used in the 3D printer are put into the 3D printer, and the monochrome FDM
printer is selected for printing according to requirements. Produced, and finally made a solid portrait of a solid
face, the same color as the three-dimensional imaging.
GH ' * OMA
HANDBOOK OF
OPTICAL FILTERS
FOR FLUORESCENCE
MICROSCOPY
by JAY REICHMAN
COPYRIGHT © 1994,1998 CHROMA TECHNOLOGY CORP. All or parts of this handbook may
be freer copied. Chroma Technology Corp. requests appropriate attribution.
Fluorescence microscopy requires optical filters that AN INTRODUCTION TO Page
have demanding spectral and physical characteris- FLUORESCENCE M|CROSCOPY 3
tics. These performance Irequirements can vary greatly Excitation end emission spectra
depend1ng on the spemñc type of mlcroscope and the . .
. . . . . . Bnghtness of the fluorescence s¡gnal
spemñc apphcat10n. Although they are relat1vely mmor .
components Of a complete microscope system, the ben- The fluorescence m¡eroscope .
efits of having 0ptimally designed filters can be quite Types ºf ñ|ters used fluorescence m|croscopy
dramatic, so it is useful to have a working knowledge of The evolution of the fluorescence microscope
the principles of optical filtering as applied to fluores-
cence microscopy. A GENERAL DISCUSSION OF OPTICAL
This guide is a compilation of the principles and kn0w- FILTERS 11
how that the engineers at Chroma Technology Corp. Termmology
use to design filters for a variety Of fluorescence micr0- Available products
scopes and applications, including wide-field micr0- Colored ñ|ter glass
se0peís, confocgl mÍCFOSCOFCS, Í1hdlaptllalications involging Thin-film coatings
51mu taneous etect10n 0 mu Up 6 uorescent pro es. A t _ t' I fi|t r
Also included is information on the terms used to de- cous o Op Ica eS
Íf53ºíf£3íºáf íiºlíííºí£iííf¿113£2?1%;Í£í£?3£¿
-
nucroscope.
DESIGNING FILTERS FOR
FLUORESCENCE MICROSCOPY 17
_ k _h 1 f h Image Contrast
Fmelly, the handh00 ends vy1t a g ossary 0 termst at Fluorescence spectra
are 1tahclzed or 1n boldface 1n the text. ,
L|ght sources
For more in-depth informati0h ah0ut the phy-si-cs and Detectors
chermstry Of fluorescence, appheat10ns fot spemflc ñue- Beamsplitters
rescth probes, sample-preparat10n techmques, and rm- 0 t' I I't
croscope 0ptics, please refer to the various texts devoted p Ica gua ' y .
to these subjects. One useful and readin available re- Optical quahty parameters
source is the literature on fluorescence microscopy and Optical quality tequitemettts fer wtde-field
microscope alignment published by the microscope m|croscopes W|th Kohler Illum|natlon
manufacturers.
FILTERS FOR CONFOCAL MICROSCOPY 25
ABOUT CHROMA TECHNOLOGY CORP. Optical quality requirements
Employee-owned Chroma Technology Corp. Specializes Nipkow-disk scanning
in the design and manufacture of optical ñlters and coat- Laser scanning
infírs for apphcati0ne th;1t re(l]-uire e3tremelpreeisien in Spectral requirements
co or separat10n, 0pt10a qua1ty, an 51gna pur1ty. Nipkow-disk scanning
low-light-level fluorescence microscopy and cyt0metry Laser scanning
spectr0graphic imaging in optical astr0n0my FILTERS FOR MULTIPLE PROBE
laser-based 1nstrumentat10n APPLICAT|ON S 29
Raman spectroscopy REFERENCES 30
Our coating lab and 0ptics shop are integrated into a
single facility operated by a staff with decades of experi- _
ence in both coating design and optical fabricati0n. We GLOSSARY G 1
are dedicated to providing the Optimum cost-effective
solution to your filtering requirements. In most cases
our staff will offer, at no extra charge, expert technical
assistance in the design of your optical system and selec-
tion of suitable filtering components.
© 1997, 1994 Chroma Technology Corp. An Employee-Owned Company 72 Cotton Mill Hill, Unit A-9, Brattleboro, Vermont 05301 USA
Telephone: 800 / 8-CHROMA or 802 / 257-1800 Fax: 802 / 257-9400 E-mail: sales©chroma.com
(BLANKPAGE)
2 CHROMA TECHNOLOGY CORP.

Fluorescence is a molecular phenomenon in which a substance absorbs AN INTRODUCTION
light of some color and almost instantaneously1 radiates light of another TO FLUORESCENCE
color, one Of lower energy and thus longer wavelength. This process is known
as excitation and emission. Many substances, both organic and n0n-or- MICROSCOPY
ganic, exhibit some fluorescence. In the early days Of fluorescence micros-
copy (at the turn of the century) microscopists looked at this primary
fluorescence, or aut0fluorescence, but now many dyes have been developed
that have very bright fluorescence and are used to selectiver stain parts of a
specimen. This method is called secondary or indirectfluorescence. These
dyes are called fluor0chr0mes, and when conjugath to other organically
active substances, such as antib0dies and nucleic acids, they are calledfluo-
rescent probes 0rfluor0phores. (These various terms are often used inter-
changeably.) There are now ñuorochromes that have characteristic peak
emissi0ns in the near-infrared as well as the blue, green, orange, and red
colors of the spectrum. When indirect fluorescence via ñuorochr0mes is
used, the autoñuorescence Of a sample is generally considered undesirable: it
is often the major source of unwanted light in a microscope image.
EXCITATION AND EMISSION SPECTRA

Figure 1 shows a typical spectrum of the excitation and emission of a ñuor0-
chrome. These spectra are generated by an instrument called a
spectr0jluorimeter, which is comprised Of two spectrometers: an illuminating
spectr0meter and an analyzing spectrometer. First the dye sample is strongly
illuminated by a color of light that is found to cause some fluorescence. A
spectrum Of the fluorescent emission is obtained by scanning with the ana-
lyzing spectr0meter using this fixed illuminati0n color. The analyzer is then
fixed at the brightest emission color, and a spectrum of the excitation is
obtained by scanning with the illuminating spectr0meter and measuring the
variation in emission intensity at this fixed wavelength. For the purpose of
designing filters, these spectra are normalized to a scale of relative intensity.
100
0300 400 500 600 700 800 900 FIGURE 1

Wave| g ) Generic excitation and emission
spectra for a fluorescent dye.
1 The time it takes for a molecule to fluoresce is on the order of nanoseconds (10-9
seconds). Phosphorescence is another photoluminescence phenomenon, with a lifetime
on the order of milliseconds to minutes.
BRATTLEBORO VERMONT 05301 USA 3

These color spectra are described quantitativer by wavelength of light. The
most common wavelength unit for describing fluorescence spectra is the
nan0meter (nm). The colors of the visible spectrum can be broken up into
the approximate wavelength values (Figure 2):
violet and indigo 400 to 450 nm
blue and aqua 450 to 500 nm
green 500 to 570 nm
yellow and orange 570 to 610 nm
red 610 to approximately 750 nm
NEAR uv v GREEN Y DARK NEAR IR >

| E RED
0 L
¡_ L
TE 61
0
R
A
N
G
E
FIG URE 2
Color regions ºf the spectrum. 320 400 450 500 570 61 o 670 750
Wavelength (nm)
On the short-wavelength end of the visible spectrum is the near ultraviolet

(near-UV) band from 320 to 400 nm, and on the long-wavelength end is the
near infrared (near-IR) band from 750 to approximately 2500 nm. The
broad band of light from 320 to 2500 nm marks the limits of transparency of
crown glass and window glass, and this is the band most often used in
fluorescence mícroscopy. Some applications, especially in organic chemis-
try, utilize excitation light in mid-ultraviolet band (190 to 320 nm), but spe-
cial UV-transparent illumination optics must be used.
There are several general characteristics of fluorescence spectra that pertain
to fluorescence mícroscopy and ñlter design. First, although some substances
have very broad spectra of excitation and emission, most ñuorochromes
have well-deñned hands of excitation and emission. The spectra of Figure 1
are a typical example. The difference in wavelength between the peaks of
these bands is referred to as the Stokes shift. Second, although the overall
intensity of emission varies with excitation wavelength, the spectral distribu-
tion of emitted light is larger independent of the excitation wavelength.2
Third, the excitation and emission of a fluorochrome can shift with changes
in cellular environment, such as pH level, dye concentration, and when
conjugated to other substances. Several dyes (FURA-2 and Indo-1, for
example) are useful expressly because they have large shifts in their excita-
tion or emission spectra with changes in concentration of ions such as
2 The emission spectrum might change shape" to some extent, but this is an
insignificant effect for most applications. See Lakowicz (1983) for an in-depth
description of the mechanism of fluorescence.

H+ (pH level), Ca2+, and Na+. Lastly, there are photochemical reactions that
cause the fluorescence efficiency of a dye to decrease with time, an effect
called photobleaching 0rfading.
BRIGHTNESS OF THE FLUORESCENCE SIGNAL

Several factors influence the amount of fluorescence emitted by a stained
specimen with a given amount of excitation intensity. These include 1) the
dye concentration within stained sections Of the specimen, and the thickness
of the Specimen; 2) the extinction coefñcient of the dye; 3) the quantum
efficiency of the dye; and, of course, 4) the amount of stained material
actually present within the field of view of the microscope.
The extincti0n coefficient tells us how much of the incident light will be
absorbed by a given dye concentration and specimen thickness, and reflects
the wavelength-dependent absorpti0n characteristics indicated by the excita-
tion spectrum of the fluorochr0me. Although many of the ñuorochr0mes
have high extincti0n coefficients at peak excitation wavelengths, practical
sample preparation techniques often limit the maximum concentration al-
lowed in the sample, thus reducing the overall amount of light actually ab-
sorbed by the stained specimen.
The quantum efficiency, which is the ratio of light energy absorbed to ñu0-
rescence emitted, determines how much Of this absorbed light energy will be
converted to fluorescence. The most efficient common fluorochr0mes have
a quantum efficiency of approximately 0.3, but the actual value can be
reduced by processes known as quenching, one Of which is photobleaching.
The combination of these factors, in addition to the fact that many speci-
mens have very small amounts of stained material in the observed field of
view, gives a ratio Of emitted fluorescence intensity to excitation light inten-
sity in a typical application Of between 10 4 (for very highly fluorescent
samples) and 106. Current techniques (e.g. fluorescence in situ hybridiza-
tion), which utilize minute amounts of fluorescent material, might have ra-
tios as low as 10 9 or 10-10.
Thus, in order to see the fluorescent image with adequate contrast, the
fluorescence microscope must be able to attenuate the excitation light by as
much as 1011 (for very weak fluorescence) without diminishing the fluores-
cence signal. How does the fluorescence microscope correct for this
imbalance? Optical filters are indeed essential components, but the inherent
configuration Of the fluorescence microscope also contributes greatly to the
filterng process.

THE FLUORESCENCE MICROSCOPE
Figure 3 is a schematic diagram of a typical epijluorescence microscope,
which uses incident-light (i.e., episcopic) illumination. This is the most com-
mon type Of fluorescence microscope. Its most important feature is that by
illuminating with incidth light, it need only filter out excitation light scatter-
ing back from the specimen or reñecting from glass surfaces. The use of
high-quality 0i1-immersion obj ectives (made with materials that have mini-
mal aut0ñuorescence, and using 10w-ñuorescence Oil) eliminates surface re-
ñecti0ns, which can reduce the level of back-scattered light to as little as 1/
100 of the incident light. In addition, the dichr0ic beamsplitter, which re-
flects the excitation light into the objective, filters out the back-scattered
excitation light by another factor of 10 to 500. (The design of these
beamsplitters is described below.)
FIG URE 3
Schematic of a wide-field
epiflu_orescence microscope, . Eyepiece
showmg the separate opt¡cal paths
for illuminataing the specimen and ?
imaging the specimen: º
illumination path , , , , , , , , , xX¿
imaging path * , , f , , * <í| .
eat filter
Filter slider ,
FIUOI'GSCGFICG L=JV
filter cube V/[I . I º , ,
B 7 7 / ,
Field Aperture Filter

diaphragm diaphragm slider
. Collctor Arc Iamp

lenses
¡ Objective
?
Specimen
An epiñuorescence microscope using Oil immersi0n, but without any filters

other than a good dichr0ic beamsplitter, can reduce the amount of observ-
able excitation light relative to observed fluorescence to levels ranging from
1 (for very bright ñuorescence) to 105 or 106 (for very weak ñuorescence.)
If one wants to achieve a background of, say, 0ne-tenth of the fluorescence
image, then additional filters in the system are needed to reduce the 0h-
served excitation light by as much as 106 or 107 (for weakly fluorescng
specimens), and still transmit almost all Of the available fluorescence signal.
F0rtunately, there are filter technologies available (described in the section
beginning on page 13) that are able to meet these stringth requirements.
TYPES OF FILTERS USED IN FLUORESCENCE MICROSCOPY

The primary filtering element in the epiñuorescence microscope is the set of
three filters housed in the fluorescence filter cube, also called the filter
block : the excitation filter, the emission filter, and the dichroic beamsplitter.

A typical filter cube is illustrated schematically in Figure 4. Figure 4
_ _ _ _ _ Schematic of a fluorescence
1) The exc1tat10n f11ter (also called the exczter) transm1ts only those wave- filter cube
lengths of the illumination light that efficiently excite a specific dye. Common . .
filter blocks are named after the type of excitation filter: o I E' 'st¡.'t%?
CU ar
UV or U Ultraviolet excitation for dyes such as DAPI and Beam59"ttef

H0echst 33342 Excitation
Filter
B Blue excitation for FITC and relateted dyes
G Green excitation for TRITC, Texas Red©, etc. Source
Although shortpass filter designs were used in the past, bandpass filter de-
signs are now used almost exclusively.
2) The emission filter (also called the barrierfilter or emitter) attenuates
a110f the light transmitted by the excitation filter and very efficiently trans- Objective
mits any fluorescence emitted by the specimen. This light is always 0f10nger
wavelength (more to the red) than the excitation color. These can be either
bandpass filters 0r10ngpass filters. Common barrier filter colors are blue or
pale yellow in the U-block; green or deep yellow in the B-block; and orange
or red in the G-block.
3) The dichroic beamsplitter (also called the dichroic mirror or dichro

matic beamsplitter 3) is a thin piece of coated glass set at a 45 degree angle
to the optical path of the microscope. This coating has the unique ability to
reflect one color, the excitation light, but transmit another color, the emitted
fluorescence. Current dichroic beamsplitters achieve this with great efficiency,
i.e., with greater than 90% reñectivity of the excitation along with approxi-
mately 90% transmission of the emission. This is a great improvement over
the traditional gray half-silvered mirror, which reflects only 50% and trans-
mits only 50%, giving only about 25% efficiency. The glass (called the sub
strate) is usually composed of a material with low autoñuorescence such as
UV-grade fused silica.
Most microscopes have a slider or turret that can hold from two to four
individual filter cuhes. It must be noted that the filters in each cube are a
matched set, and one should avoid mixing filters and beamsplitters
unless the complete spectra] characteristics of each filter component
are known.
Other optical filters can also be found in fluorescence microscopes:
1) A heatfilter, also called a hot mirror, is incorporated into the illuminator

collector 0ptics of most but not all microscopes. It attenuates infrared light
(typically wavelengths longer than 800 nm) but transmits most of the visible
light.
2) Neutral density filters, usually housed in a filter slider or filter wheel
between the collector and the aperture diaphragm, are used to control the
intensity of illumination.
3 The term dichroic" is also used to describe a type of crystal or other material that
selectively absorbe light depending on the polarization state of the light. (Polaroid©
plastic film polarizer is the most common example.) To avoid confusion, the term
"dichromatic" is sometimes used.

3) Filters used for techniques other than fluorescence, such as color filters
for transmitted-light microscopy and linear polarizng filters for polarized
light microscopy, are sometimes installed.
THE EVOLUTION OF THE FLUORESCENCE MICROSCOPE4

The basic configuration of the modern fluorescence microscope described
above is the result Of almost one hundred years Of development and innova-
tion. By looking at its development over the years, one can gain a better
understanding of the function of these various components.
The first fluorescence microscopes achieved adequate separation of excita-
tion and emission by exciting the specimen with invisible ultraviolet light.
This minimized the need for barrier filters.5 One of these turn-0f-the-
century microscopes used for its light source a bu1ky and hazardous 2000 W
iron arc lamp ñltered by a combination Woodºs solution (nitrosodimethylaniline
dye) in gelatin, a chamber Of liquid copper sulfate, and blue-violet colored
filter glass. This first excitation filter produced a wide band Of near-UV light
with relativer little visible light, enabling the microscopist to observe the
inherent primary fluorescence of specimens. Microscopists were aided by
the fact that most substances will fluoresce readin when excited by UV
light. In 1914, ñuorochromes were first used with this type of microscope
to selectively stain different parts Of cells, the first utilization of secondary
fluorescence.6
These first fluorescence microscopes, illustrated schematically in Figure 5,
used diasc0pic (i.e., transmitted light) illumination. Both brightfield and
darkfield 0i1-immersi0n condensers were used, but each had certain impor-
tant disadvantages. With the brightfield condenser, the maximum intensity
of illumination was severer limited by the capabilities of the optical filters
that were available at the time. The darkfield condenser, which directed the
Specimen GIass/Liquid
Slide Exciftation Quartz
FIGURE 5 ºbjective 1 Condenser F'Itexr Cºt"eerfstºr
t;tf:::f¿:3:¿¿:£tt:311%3£ºº AI::_::_:_,_ [[ o' " 53:33
. . _ Eyepiece
(After Kasten, 1989.) -¿ "
_
excitation light into a cone 0f1ight at 0h1ique angles, prevented most of the
excitation light from entering the objective lens, thus reducing the demands
on the optical filters. However, the efficiency of the illumination was greatly
reduced, and the objective lens required a smaller numerical aperture, which
resulted in a further reduction in brightness as well as lower resolution.7
2Wettim1istalwmºmmtm
5 The first barrier filter to be used was a pale yellow coverslip, which protected the eye
from hazardous radiation, but some of the early fluorescence microscopes might have
Iacked even this.
6 Several fluorescent dyes were synthesized in the eighteenth century for other
purposes, including use as chemotherapeutic drugs that stained parasitic organisms
and sensitized them to damaging rays.
7 Abramowitz (1993).

The most important advance in fluorescence microscopy was the develop-
ment Of episcopic illumination for fluorescence microscopes in 1929. Episcopic
illumination was first utilized to observe the fluorescence Of bulk and 0paque
specimens. These first epiñuorescence microscopes probably used half-si1-
vered mirrors for the beamsplitter, with a maximum overall efficiency of
25%, but important advantages were 1) the ability to use the high numerical
aperture objective as the condenser, thus achieving greater brightness; 2) the
fact that the intensity of excitation light that reflects back into an 0i1-immer-
sion objective is, as discussed above, roughly 1% of the incidth light;8 and
3) ease of alignment. The introduction of dichr0ic beamsplitters by Brumberg
in 1948, and their subsequent commercialization by Ploem in 1967, im-
proved the efficiency of the beamsplitter to nearly 100% and further im-
proved the filtering effect of the microscope.
The following important technical advances, together with the advent of the
epiñuorescence microscope, helped revoluti0nize fluorescence microscopy
during this historical period: 1) the development of compact mercury-vapor
and xenon arc 1amps (1935); 2) advances in the manufacture of colored filter
glasses, which enabled the use of ñu0rochromes that were efficiently excited
by visible light (thus allowing, for example, the use of simple tungsten fila-
mth light sources); 3) advances in microscope objective design; and 4) the
introduction of anti-reñecti0n coatings for microscope 0ptics (c. 1940).
These developments were, of course, driven by great advances in the bio-
logical and biomedical sciences over the years, and revoluti0nary develop-
ments have continued with the advent of ultrasensitive cameras,9 laser
illumination, confocal microscopy, digital image processing, the continuing
development of hundreds of fluorochr0mes and fluorescent probes, and, of
course, great improvements in the capabilities of optical filters.
8 Assuming nonmetallic specimens.

9 Inoué (1986) is an excellent text detailing the use of video imaging and microscopes
in general.

(BLANKPAGE)

B ef0re describing in detail the design of optical filters for fluorescence mi A GENERAL
croscopy, it is worthwhile to introduce some Of the terms used to specify
filter performance, and describe the characteristics of available products. 3IPS1%gÍEIFOIETOEFRS
TERMINOLOGY
Although color designati0ns such as U, B, and G are often adequate for de-
scribing the basic filter sets, it is useful to be familiar with the terms used for
more precise descripti0ns 0ffi1ters, especially when dealing with special sets
for unusual dyes and probes. The most common unit for describing filter
performance is the wavelength 0f1ight in nanometers, the same as for ñu0r0-
chrome spectra described previously. Note that the perceived color of a filter
depends on the bandwidth (described below) as well as specific wavelength
designati0n. This is especially noticeable when looking through filters in the
range of 550 to 590 nm: a filter with a narr0w band will look pale green while
a filter with a wide band, especially a longpass filter, will look yellowish or
even bright orange.
Some of the important terms used to describe the spectral performance of

optical filters are defined below. Please refer to the illustrati0ns in Figures 6
through 9.
1) Bandpass filters are denoted by their center wavelength (CWL) and band 9 ?º
width (FWHM). 10 The center wavelength is the arithmetic mean Of the wave- A) Nomenclature fºg¿;igg£:.f&gg
lengths at 50% 0fpeak transmission. The FWHM is the bandwidth at 50% of B) Nomenclature fºr blocking
peak transmission.
2) Longpass and shortpass cut-0n filters (LP and SP)

are den0ted by their cut-0n or cut-0ff wavelengths at A) LP Bandpass sp
50% of peak transmission. LP or SP filters that have a 100 _ _ _ Peak %
very sharp slope (see next page) are often called edgejíl Peak CWL Avg. %T
ters. The average transmission is calculated over the useful
transmission region Ofthe filter, rather than over the entire
spectrum. (Please note that the use Of terms highpass %T 50% 50%
and lowpass are discouraged because they more accu- º t'º FWHM % % CUt'ºff
rater describe frequency rather than wavelength.)
3) The attenuation level, also called blocking level, and 0
attenuation range, also called blocking range, are n0r- Wave|ength )
mally defined in units of optical density (OD):
OD : log(T) or OD : log(%T / 100) B) (Bandpass)

Example: OD 4.5 = 3 X 10 5 T (0.003 %T) º
Optical density uses the same logarithmic units as the .
quantity absorbance, which is a measure Of absorption, B.Ock¡ng
but filters can attenuate light in various ways other than O-D- B|0%k¡ng Rangº
absorpti0n. For example, thin-film interference filters Level
block primarily by reflection, and acoust0-optical filters
block by diffracti0n. Therefore, the term optical den- Peak Attenuation
sity is more precise. (Both of these filter products are 6
described in detail in the section beginning on page 13.) Wavelength (nm)
10 Full Width at Half of Maximum Transmission

o 7? * ?7 A term related to attenuation level is cross-talk (Figure
7), which describes the minimum attenuation level (over
a specific range) of two filters placed together in series.
This value is important when matching excitation filters
with emission filters for a fluorescence filter set.
0D 4) The term slope describes the sharpness of the transi-
Crºss»talk Level tion from transmission to blocking. Figure 8 illustrates
two sets of filters with the same bandwidth or cut-0n
8 point, but different slope. In the Figure, note that al-
450 WºVº'º"ºth ("m) 600 though the bandpass filters look very similar on a 100%
transmission scale, the slopes as indicated on the optical
FIGURE 7 density scale are significantly different. Slope can be
Cro_ss-talk level of two filters in specified by statng the wavelength at which a particular
senes' filter must have a specified blocking level.
5) The angle of incidence (AOI) is the angle between
A) the optical axis of the incident light and the axis normal
lºº ' º* ' to the surface Of the filter, as illustrated in Figure 9.
Most filters are designed to be used at zero degrees
angle of incidence, called normal incidence, but for
' beamsplitter coatings the usual angle is 45 degrees. It
% ' should be noted that most types of filters, such as thin-
film interference coatings and acoust0-0ptical crystal
devices, are angle-sensitive the characteristic per-
0 formance changes with angle. (These products are de-
450 Waveleng ) 600 scribed in greater detail in the next section.) If a filter or
B) beamsplitter is to be used at any angle other than the
0 f' 'N usual zero or 45 degree angle, it must be specified ex-
plicitly.
One consequence of angle sensitivity is that the half-
) cone angle of the incident light might need to be speci-
fied if the filter is to be used in a converging or diverging
beam (Figure 10). The half cone angle can also he de-
scribed in terms Of the f-number, or the numerical ap
6 erture (NA) of the light beam, which equals the sine of
45 Wavelength (nm) 600 the half-cone angle.
6) Dichr0ic beamsplitters (and, in fact, any thin-film
FIGURE 8 interference coating that is used at n0n-normal angle of
Filter S?ÍS With varying S|0PF, . incidence) will cause some amount of polarization, the
shown A) percent transm¡ss¡on, . . .
and B) in optical density. exact effect vary1ng great1y w1th wavelength and w1th
the pa1ticular design. Some relevant terms are illustrated
in Figure 9: P plane (also called TM mode,
i.e., transverse magnetic ) is the component Of the light
beam's electric field that is parallel to the plane of inci-
c¡de No,ma| dence of the beamsplitter, and S plane (TE mode,
X / i.e., transverse electric ) is the component of the light
Aº' Plane of incidence
, ,
gzglealgel / B ººmsp ¡'n' * ºººt''"º FIGURE 9
E EÍ,'Í;M glass SUb3trate Schematic illustration of terms used to describe polariza-
tion. The normal axis is the axis perpendicular to the
surface of the coating, and the plane of incidence is
defined by the normal axis and the direction vector of the
incident light beam.

beam's electric field that is perpendicular to the plane of incidence of the Filter
beamsplitter. The polarizng effect Of a typical dichroic beamsplitter is illus- HaIf-cone angle
trated in Figure 11. T
Light cone
AVAILABLE PRODUCTS í
The two main types Of filter technology used in fluorescence analysis are
colored filter glass and thin-film coatings. In addition, acoust0-optical tun- .
. . . . . . . . F|gure 10
able f11ters are f1nd1ng 1ncreased use1n spe01al apphcat10ns. These products .
. . . . . Illustration of haIf-cone angle
are descr1bed below. Other products, such as holograph10 f11ters and 11qu1d- ºf divergent or convergent
crystal tunable filters, are available, but they are used infrequently in ñu0res- incident light.
cence microscopy.
Colored F||ter Glass

.
1
100
Colored filter glass, also called absorption glass, is the _ _

_ _ _ P plane polanzat¡on
most w1dely used type 0ff11ter1n fluorescence analy51s, Randºm pºlarizatiºn
particularly the yellow and orange sharp-cut glasses and % s.p.ane pº|a,¡zat¡ºn
black glasses that transmit the UV and absorb the
visible. Filter glass attenuates light solely by absorption,
so the spectral performance is dependent on the physi-
cal thickness of the glass. Increasing the thickness Will 0
increase the blocking level, but also reduce the peak in- 400 Wove|engm 600
band transmission (Figure 12), so an optimum thick-
ness value must be determined. Stock thicknesses FIGURE 11
offered by the glass manufacturers represent a thick- Polarizing effect of atypical dichroic mirror. This
- - particular coating is designed for reflecting the 488 nm
ness value that IS Ityplcal for the general uses ºf the Iinearly polarized argon-ion laser line in the S-plane.
glass, but other th10knesses m1ght be better for a spe-
ciñc application.
. . A
Followmg are some advantages of f11ter glass: ) 100
1) It is relatively inexpensive.
. . . . 1
2) It1$ stable and 10ng-hved under normal cond1t10ns.11 2 23
3) Its spectral characteristics are independent of angle 9
of incidence, except for slight changes due to increased
effective thickness.
Disadvantages Of filter glass include the following: o
_ _ _ _ 350 Wavelength (nm) 800
1) There15 a hm1ted select10n of glasses. B)
2) The bandpass types have poor slope and often low 6
peak transmittance.
3) There is less flexibility in the specification of filter 2 mm
thickness because of the dependence of spectral per- 0.0. 1 mm X
formance on thickness.
4) Most of the longpass filter glasses have high auto-
ñu0rescence. 0
. . . I h 800
5) Smce absorpt10n converts most of the rad1ant en- 350 wave engt (nm)
" Some minor exceptions are 1) sharp-cut Iongpass filter FIGURE 12

glasses have a shift in cut-on of approximately 0.1 to 0.15 nm/ Spectra ºf near-IR blocking glass (Schott© BG-39) at1 mm
ºC temperature change; and 2) some types of fllter glass can be and 2 mm thickness, shown in
affected by unusual environments such as intense UV radiation A) percent internal transmission and B) optical density.
("solarization") or high humidity. (Schott Glasswerke catalogue.) (From Schott Glasswerke catalogue.)

ergy into heat, untempered filter glass might crack un-
NOT TO SCALE !!! der conditions of intense illumination.
Iní|;hñnt 3223fá3d$º£1erference Included in the category of filter glass are polymer-

¡ based filters, which are sometimes used as longpass
.a ¡ barrier filters because they have low autoñuorescence
!:¿º26. compared te an equivalent filter glass, ahd a type of
=ag&9_ >Th¡n_f¡|m neutral-denstty glase (not to be confused w1th th1n-f11m
=u-" layers neutral-den51ty coat1ngs descr1bed below).
Thickness '
|_ Thin-Film Coatings
* _ Transmitted light, . . . . .
modified by ¡ erference Two w1de1y used categ0r1es of th1n-f11m coat1ng
are: 1) metallic coatings for making fully reñective mir-
rors and neutral-density filters, and 2) thin film inter
FIG RE ference coatings, which are the main component of
Sch2mat12 illustration ºf a thin-film interference filters. The main advantage of thin-film interference coatings is
interference coating the tremendous ñexibility Of performance inherent in the way they work. As
shown in Figure 13, interference coatings are composed of a stack of micro-
scopically thin layers Of material, each with a thickness on the order of a
wavelength 0f1ight (usually around a quarter of a wave1ength of light
approximately 1/ 10,000 of a millimeter in thickness). Although each
material is intrinsically colorless, the reflections created at each interface
between the layers combine through wave interference to selectively reflect
some wavelengths 0f1ight and transmit others. A common natural example
of thin-film interference is the formation of swirls of color on a soap bubble.
Interference occurs between the reñecti0ns from the inner and 0uter sur-
faces of the bubble, and the colors follow contours of constant thickness
within the single layer of soap.12
Almost any filter type can be designed using interference coatings, including
bandpass, shortpass, and longpass filters, and dichroic beamsplitters. By
adjusting the number of layers in the stack and the thickness of each layer,
one can control to high precision the nominal wavelength, the bandwidth,
and the blocking level. One can also create filters with
100 greater complexity than the standard bandpass, 10ngpass,
or shortpass. For example, filters with multiple bands,
illustrated in Figure 14, are now produced for com-
mercial use and are valuable tools in fluorescence mi-
%T croscopy.
There are several limitati0ns to thin-film interference
coatings, including the following:
o 1) The characteristic blocking performance only holds
380 400 500 600 700 within a finite wavelength range. However, additional
wave'e"gth ("m) components can be added to the filter, such as wide-
FIGURE 14 band blockers and the filter glass mentioned above, in
Spectra ºf a tripIe-band filter order to extend this range. The addition of these blocking components can
designed for DAPI, FITC, and reduce the peak transmission of the finished filter and increase the physical
12232¿:$? gg :;%º&º¿gggg&?º thickness. An example Of a filter with no added blocking (often calle<1 an un-
blocked f11ter), and the same f11ter blocked w1th added components, IS shown
12 But unlike a soap bubble, the optical thin-films are of solid material (either
polycrystalline or amorphous), and the coating layers are extremely uniform in
thickness.

in Figure 15. A)
. . . . . . 100 » , < Fi Iter
2) The coat1ng mater1als used are 11m1ted1nthe1r range / * x,
of transparency. Outside of this range, coatings become / 3 ,B|ocker
highly ahsorhing rather than highly transmissive 0r re- *,
. . . . < Glass
ñect1ve. Some of the coatmg mater1als that are 1deal for % tx
visible filters have excessive absorption in the UV, so x
materials with different and less than ideal characteris- X
tics must be used for UV filters. As a result, UV filters X
. . - 0
tenc1te have more 11m1ted performance and less de51gn 400 Waveleng 1000
ñex1b1hty. Another consequence IS that one cannot a1- B)
ways calculate the reñectance from a transmission spec- lºº
trum by assuming zero absorption in the wavelength
range shown in the spectrum. Figure 16 is an example
of a dichroic beamsplitter designed for high reñectivity
in the visible only. The drop in transmission in the UV %
is a result of absorption instead Of reflection. In gen-
eral, one cannot assume that a beamsplitter designed to
reflect the visible will also reflect the UV. 0
3) As noted on page 12, interference coatings are sensi- Wºvº'ºnºth < ) 1000
tive to angle of incidence. As the angle is increased, the FIGURE 15 (above)
spectral characteristics of the coating shift_ to shorter . .
_ _ 3 1 An example of a blpcked fl|ter. A) spectra ef an ur_|blocked
wavelengths, 1.e., the Spectrum ls blue-shlfted. In filter and blockmg components, mcludmg aw¡de-band
addition th 1 'Zin ff f 'n 1' 1 "blocker" and infrared-blocking blue filter glass; and B) the
. . 61.30 am .g e ect0 coat1 g-s ateb 1que ang es spectrum of the blocked filter. The blocking range in the
0f1n01dence 15 unde51rahlem most apphcat10ns. Although infrared (not shown) is determined by the range of the blue
the coating designer can minimize the polarization, it glass,approx¡mately1.2 m¡crons.
ca-n-not be ehmmated ent1re1y. Th1$ effect15, however, FIGURE 16 (below)
Ut1hsz advantageously m some spe01al apphcat10ns. Reflectivity vs. absorptance. This dichroic beamsplitter
sample has high absorption in the UV.
Acousto-Opt¡cal Fllters
The acoust0-0ptical tunable f11ter (AOTF), shown sche- 100
matically in Figure 17, is most often used for filtering Absggº¿;gg
excitation light, especially laser excitation. This filter
works by setting up radio-frequency acoustical vibra- X
ti0ns in an appropriate crystal and creating, in effect, a % % Rºf'ººtiº"
bulk transm15510n d1fftact10n grat1ng. By very1ng the %Transmissíºn
frequency, one can rap1d1y tune the f11ter to d1ffract out
any wavelength of light within the useful range, with
high precision. A typical AOTF will accept incident light 0
with a maximum half-cone angle of approximately 5 ººº Wºvº'º gth ( m) ººº
degrees. The AOTF is an electronically controlled de-
vice that uses an external control unit.
Te0¡ Acoustic
Advantages of the AOTF include: CW ? "

White (+) Di"racted Beam
1) the ability to change to any wavelength within microseconds, perform º' -% e

wavelength scanning, and generate multiple-band ñltering by mixing multiple 3333 &&
radio frequencies; and Wº"º ,,,, |
2) the ability to rapidly vary the intensity by changing the amplitude Of the T¿¿u¡¿.¿r (D 333?
. . .
acoust10 v1brat10ns. RF5wÍ ..,Mo 2ed
Vsnau onoc romatic
FIG U RE 1 7
_
13 The effective physical thickness of the thin-film coating does indeed increase with Schematic representation of an
increasing angle of incidence, but the difference in path length of the interfering acousto-optical tunable filter.
reflected light rays decreases. (From Brimrose Corp.)

Some disadvantages include:
1) limited FWHM (approximately 2 nm in the visible), which restricts the
available light output from white light sources when the AOTF is used as an
excitation filter;
2) limited physical dimensions such as a small aperture (approximately 10
mm or less) and a large overall thickness of approximately 25 mm; and
3) linearly polarized output, giving a maximum of 50% T when using unp0-
larized incident light.
Liquid Crystal Tunable Filters*

The liquid crystal tunable filter (LCTF) is another electronically controlled
device that is finding increasing use as an emission filter, because it works
on a principle that enables imaging-quality filtering with an ample clear
aperture and an in-line optical path. At the heart of the LCTF are a series of
waveplates, each composed of a layer Of birefringent material paired with a
liquid crystal layer, and sandwiched between linear polarizers. The birefrin-
gence Of the liquid crystal layer, and thus the total magnitude Of the waveplate,
is fine-tuned by varying the voltage applied to transparth conductive coat-
ings adj acent to the liquid crystal layer.
Brieñy, the birefringence Of the waveplate induces a wavelength-dependent
rotati0n of the incoming polarized light. The second polarizer then attenu-
ates the polarized and rotated light to varying degree, in effect converting
the rotati0n into an amplitude variation which is also wavelength-depen-
dent. The LCTF designer is able to control filtering parameters by varying
structural aspects such as the number of waveplates in series and the hire-
fringence qualities of each waveplate.
Characteristics Of LCTFS include:
1) a speed of wavelength selection on the order of milli-seconds;
2) no wavelength-dependent image-shift;
3) variable attenuati0n capabilities; and
4) a choice Of bandwidths (FWHM), spectral range of tunability, and block-
ing level, although these parameters are somewhat interdependent.
LCTFS are polarizng optical components, so the maximum peak transmis-
sion for unpolarized light is fifty percent. Existing devices can have signifi-
cantly less than fifty percent, depending on wavelength and blocking level.
However, the use of special polarizng beamsplitters in an epi-ñuorescence
microscope can mitigate the overall effect of these losses. The maximum
blocking level commercially available is around 105.

The primary goal of filter design for fluorescence microscopy is to create DESIGNING
filter sets (typically an excitation filter, emission filter, and dichroic FI LTERS FOR
beamsplitter) that maximize image contrast and maintain image quality.
Image contrast is a combination of several factors: 1) the absolute brightness B%gggggggºCE
of the image; 2) the brightness Of the fluorescng substance relative to the
background, known as signal t0 noise (SW ); and 3) to a lesser extent in the
case Of visual or photographic 0hservati0n, the color balance as perceived by
the eye. Filters must have high throughput (i.e., wide bandwidth combined
with high transmittance), as well as low cross-ta1k, because no amount of
improvement in S/N will improve the image contrast if the image is not bright
enough to be adequater detected. In addition, the best way to achieve maxi-
mum contrast often depends on the specific application or technique, so the
filter designer should have a conceptual understanding Of these various appli-
cations. How the filter designer incorporates the available information re-
garding a particular application is described in detail below.
Image quality is maintained by ascertaining the optical quality requirements
at each point in the microscope where a filter is inserted, and assuring that
each filter is manufactured to the correct specifications. In order to ascertain
these requirements, one must be well grounded in the fundamentals Of micro-
scope 0ptics and have an understanding of the requirements of particular
applications and techniques. This is especially true today, with the growing
number of applications that are taking advantage of the newest technologies:
1asers for both illumination and sample manipulati0n, digital image process-
ing, computer-assisted positi0ning and controls, and ultrasensitive detection
devices.
In addition, the filter designer must be aware of all the physical dimensions
required by the various microscope brands and models.
IMAGE CONTRAST
The general process by which a filter set is optimized for a particular fluoro-
chrome can be illustrated by taking as an example a specimen staíned with the
dye FITC and explaining how filters are designed for this dye.
Fluorescence spectra
The single most important parameter for designing a filter set is the spectral
characteristics of the dye, with excitation and emission spectra shown in Fig-
ure 183 (next page). If this were the only parameter to be considered, one
would illuminate the specimen using a shortpass excitation filter that trans-
mits all Ofthe excitation spectrum, and observe the fluorescence using a 10ngpass
emission filter that transmits the entire emission spectrum. A pair of filters for
FITC having these characteristics is shown in Figure 18h. These represent
ideal shortpass and longpass filters: real filters would need a wider separa-
tion between the cut-0n and cut-0ff because of slope limitati0ns of f11ters, and
the shortpass excitation f11ter would have a cut-0ffpoint somewhere in the UV.
But in a real specimen there are other considerati0ns. Many substances in the
specimen are likely to aut0ñuoresce if a shortpass excitation filter is used,
especially one that transmits UV light. Pathological tissue specimens are es-
pecially pr0ne to autoñuorescence. Also, the presence Of UV light, which has
higher intrinsic (i.e., quantum) energy, might increase the rate 0fphotobleaching
of the ñuorochr0me and/0r cause ph0t0damage to the specimen. Therefore,

FIGURE 18 C)
A) FITC excitation and emission spectra 100
B) "Ideal" filter pair for FITC, overlaid on excitation and
emission spectra / . . .
C) E:_I|tgr pair with bandpass excitation filter specific to 233333 e, Em'ss'ºn mm
D) TRITC excitation and emission spectra, overlaid on %
FITC spectra
E) Bandpass filter pair for FITC, overlaid with excitation/
emission spectra for FITC and TRITC (Chroma
Technology Corp. P/N 41001)
o . , .
300 400 500 600
Wavelength (nm)
A) D)
100 100 ,x x
1 ' ¡
TRITC Excitation ' ¡ l ,
X I ,
_ _ x xx,, 1'( ¡X _TR|_TC
Exc¡tat¡on Emission ¡ Emiss¡on
% ,, ' ') h
'6 ¡I , | X
I ¡ x
/
, l ¡ x
/ ' x x x
/ I x
/ I
o 0 " x _ __ _ _ _ _ , / /
300 400 500 600 300 400 500 600
Wavelength (nm) Wavelength (nm)
B) E)
100 100
/ Béan0pess
- - / muss¡on
Excitation F||ter Emission Filter Bandpass Exc¡tet|£
% %
o 0
300 400 500 600 300 400 500 600
Wavelength (nm) Wavelength (nm)
100 one should limit the band to a region where the FITC
365 436 excitation is at a maximum, but still wide enough to
allow adequate intensity, using a bandpass excitation fil-
546 ter as shown in Figure 18c.
4º5 215 . . . .
y 579 If a second ñuor0chr0me IS 1ncluded 1n the spec1men,
for example TRITC with excitation and emission spec-
tra as shown in Figure 18d, there is low but significant
excitation efficiency in the blue for this dye. If a longpass
o emission filter is used for the FITC, a small but notice-
300 600 900 able orange emission from TRITC might be seen. This
is usually considered an undesirable effect, especially
FIGURE 19 when imaging with a monochrome camera that does
Spectrum of a mercury arc Iamp. (Mid-UV output below not distinguish between colors. In this case, one should
3ºº " '5 "ºt ShºW"-) restrict the FITC emission filter to a narrower band (Fig-
ure 18e) that is more specific to the band of peak emis-
sion for FITC. The f11ters in Figure 18e are examples Of

Chroma Technology's 41-series HQ filters. (Note that the excitation filter
attenuates any light that could be transmitted by the leak in the emission
filter spectrum at 410 nm.)
For cyt0metric applications, where image brightness is not critical, even more
narrow bands are often used in order to maximize the selectivity between fluo-
rochromes.
Light sources A)
So far, a hypothetical light source having a equal output 100
in all colors a pure white light has been assumed. EQ$%%gfd
The most common light source for fluorescence micros- Spectrum
copy, chosen for its high brightness (known technically 5&%233[¿ EXC¡ ¡ Spect m
as luminance or radiance) in the ultraviolet and visible % X Mg9¿3ºff,5% Lamp
spectrum, is the mercury arc1amp. The spectrum Of this
light source (Figure 19) is far from continuous most
Of its light output is concentrated in a few narrow bands,
called lines, and each line is approximately 10 nm wide. 0
Most general-purpose filter sets should have excitation 300 400 Wavelength mm) 500 ººº
filters that transmit one or more of these lines, but there
can be noticeable exceptions, one Of which is illustrated B) 100
in the 0ngoing example. Figure 20 shows the effect of Standard
modifying the excitation spectrum of FITC by the 0ut- Exº"º"º" F¡'tºfx
put of a mercury arc illuminator. A wide-band excita- Excit£ggñ-Eíg
tion f11ter that included the light at 436 nm would provide %
an emission signal significantly hrighter than the filter Exc¡tat¡ºn Spectrum
that excludes this line. (A factor of 1.25 to 1.5 would be M,,,,, , gºggfifgntg
expected.) But for most specimens a reduction in overall
S/N is to be expected because the increase in noise from 0
autoñuorescence will 0utweigh the increase in ñu0res- 300 400 500 600
cence signal. However, for certain applications involv- Wºvº'ºngth ( m)
ing extremely low absolute levels Of ñu0rescence, or for
specimens in which the FITC spectrum has been blue- FIGURE ºº
shifted,14 a wide-band excitation filter that includes the A) FITC excitation spectrum ""mºdifiedº and_ mºdified by
. . . . . the mercury arc Iamp spectrum (normaluzed to 100%
436 nm hne m1ght prov1de 1mpr0ved detect10n. Note relative peak T).
that the same emission f11ter would be used regardless Of B) Modified FITC excitation spectrum overlaid with
excitation band because the emission spectrum would standard and wide-band excitation filters.
not be significantly altered.
Another light source used in fluorescence microsc0py is
the xenon arc lamp (Figure 21), which does have a rela-
tiver continuous spectrum in the visible. Xenon arc lºº 828
lamps are preferred in systems where the spectral charac-
teristics of dyes and/0r specimens are being analyzed
quantitatively, but they are not as bright as a mercury
lamp of equivalent wattage. Even in the region of FITC %
excitation (between 450 and 500 nm) where the mer-
cury 1amp is relativer weak, the xenon arc lamp is only
marginally brighter. This is primarily a result of the fact
that the light-pr0ducing arc of the xenon 1amp is about 0
twice the size of the arc in the equivalent mercury lamp, 300 600 900
14 This shift can occur, for example, under conditions of low FIGURE 21
pH values (pH less than 6) (Haugland, 1992). Spectrum ºf a xenon arc Iamp

which reduces the amount of available light that can be focused onto the
specimen using a typical microscope conf1guration. Some relevant data con-
cerning mercury and xenon arc1amps are given in Table 1. Note the differ-
ences in arc size between the lamps.
100 Detectors
488 5145 The excitation filter must be designed to block any out-
0f-band light that can be picked up by the detector. Arc
lamps and filament1amps have output throughout the
near-UV, visible, and IR, so the filter must have ad-
% 476 496 equate attenuati0n over the whole range of sensitivity
of the detector. But for laser illumination, the blocking
458 529 range of excitation filters need only cover the range of
351 tº 363 output of the laser. For example, IR blocking is not
0 required for the argon-ion laser (Figure 22.)
300 600 900
Figure 23 shows some sensitivity spectra Of important
FIGURE 22 (above) detectors. Not shown is the sensitivity of unintensified
Typical spectrum of an argon-ion laser. silicon photodiodes or CCDS, which have sensitivity to
(Data frºm Speºtra'phys'ºs Lasers, 'nº') 1100 nm, falling to zero by 1200 nm. Note that silicon
FIGURE 23 (below) detect0rs that are 1nten51f1ec1 0v1th, for example, a
. . . . . m1cr0channel plate, have sen51t1v1ty ranges sumlar to
Sens¡t¡v¡ty of var¡ous detectors. Each spectrum ¡s _ _ _ _ _ _
normalized to peak senstivity. the 1nten51f1ed v1de0 spectra shown m F1gure 23.
(Video and PMT data from Hammamatsu Corp.) _ _ _ _
In generaht15 preferable to block 0ut-0f-band 11ght w1th
. the excitation f11ter instead Of the emission f11ter for three
100 . . .
50 Intensified Videos reasons: 1) the spec1men w111be exposed to less rad1a-
x tion; 2) fewer components in the emission filters gener-
ally improve its optical imaging quality; and 3) many
% 5 X microscopes have shallow cavities for holding emission
Humº" ZÍTVS filters, so it is beneficial to eliminate components that
1 7**7777 **? add to the finished thickness. There are certain cases,
05 for example UV excitation, where the process Of extend-
0_1 ing the blocking of the excitation filter great1y reduces
300 500 700 900 the peak transmission Of the filter. In these cases itwou1d
wave'ength ("m") be appropriate to provide extended IR blocking in the
emission f11ter instead.
When doing visible photographic work, it is important
to have IR blocking because some built-in light meters
TABLE 1 . . . .
, have IR sen51t1v1ty that could affect exposure t1mes.
Data for xenon and mercury arc Iamps. Boldface entr¡es
indicate most common sizes for fluorescence
microscopes. (From Abramowitz, 1993)
RATED POWER LUMINOUS FLUX AVERAGE BRIGHTNESS ARC SIZE RATED LIFE
(watts) (lumens) (candeIa/mmº) w x h (mm) (hours)
Mercury:
HBO 50W/3 50 1300 900 0.20 X 1.35 200
HBO 100W/2 100 2200 1700 0.25 x 0.25 200
HBO 200W/2 200 10000 400 0.60 X 2.20 400
Xenon:
XBO 75W/2 75 950 400 0.25 x 0.50 400
XBO 150W/1 150 3000 150 0.50 X 2.20 1200

Beamsplitters
The final stage in the design of a filter set is to select a 100 _
dichroic beamsplitter that matches the spectra of the ex- Exº'tº' x .
citation and emission filters. Reñectance Of greater than EíítTesrphtter
90% Of the excitation light is desired, with average trans-
mission greater than 90% in the emission band. At the
relativer high angle Of incidence Of45 degrees, the coat-
ings can be highly polarizing, so the designer must work
to minimize this effect. Beamsplitters for imaging sys-
tems, such as microscopes, usually consist of one or two o
coatings applied directly onto the glass substrate, so ex- 300 400 Wavelength mm) 500 600
tra care in handling is advised. The surface that is de-
signed to face the light source and specimen is called the FIGURE 24
front surface of the beamsplitter. Determinng which is Completed fi|ter set design fºr FITC
the front surface can be difficult, so the manufacturer (Chroma Tecnology Cº"|º- PM 41001)
usually provides some type of markng to indicate the
correct orientation.
Figure 24 sh0ws a c0mp1etec1 design Ifor-a filter set for SURFACE FLATNESS
FITC, 1nclud1ng exc1tat10n f11ter, ermsswn f11ter, and
dichroic beamsplitter matched to these filters. %
W;czssst ¿
OPTICAL QUALITY
The optical quality requirements for an optical filter de-
pend very much on such factors as the type of filter (es- lí
pecially whether it is a component of the illumination Ref|ected
0ptics or the imaging/detection 0ptics), the type of mi- wavºfrºf
croscope, and the intended application. For example, TRANSMITTED WAVEFRONT DISTORTION
the optical quality specifications for an emission filter
used for quantitative image analysis with a laser scan- . % % .
ning confocal microscope are much different from those WÁ323331 % % Ivg1eS/rglgfd
for an excitation f11ter used for qualitative visual 0hser- % %
vati0n with a standard wide-field microscope. Although
specific requirements for every type of filter and every
application cannot be described here, by introducing the WEDGE (PARALLELISM)
key optical quality parameters and analyzng a few basic
microscope configurations, one can develop a set Of gen- . % %
eral guidelines that can be applied to most situations. WÁÚ%'3331 % % Deviated
% Hwavefront
Opt|cal q0ahty perameters Wedge angle ¡,

The followmg 15 a hst of 1mportant parameters used to 5,/
def1ne the overall image quality Of a f11ter or beamsplitter. /
The first three parameters are illustrated in Figure 25.
1) Surface Hatness is a measure of the deviation of the
surface Of an optical element from a perfect plane, usu- FIGURE 25
ally measured in fracti0ns or multiples Of a wavelength Illustration of the effects of surface
of visible light (usually 550 or 630 nm, but sometimes flatness, ¿123333333532?
using the CWL for a bandpass filter). The actual
wavefr0nt distorti0n that a plane wave of light under-
g0es when reflected from the surface is twice the value

of the surface flatness.15 The wavefr0nt distorti0n of light reflected off a
beamsplitter or mirror is solely determined by the surface ñatness of the re-
flecting surface, usually the front surface.
2) Transmitted wavefront distortion (TWD) is a measure of the dist0rtion
a plane wave of light underg0es when transmitted through an optical ele-
ment, also measured in fracti0ns or multiples of a wavelength of light, the
same as for surface ñatness, described above. The surface ñatness Of the 0uter
surfaces of the element and, to a lesser degree, internal structures that cause
inhomogeneity of the refractive index, combine to make up the overall TWD
of the optical element.
3) Wedge (also called parallelism) is a measure of how parallel are the 0uter
surfaces Of an optical element. Wedge is usually measured in arc-minutes or
arc-seconds Of angle. The main effect Of wedge is to induce an angular devia
tion in the direction of a light beam, causing, for example, image shifting. The
amount of angular deviati0n is about 0ne-half the wedge angle for a typical
f11ter.16 The wedge Of internal coatng surfaces, while not contributng greatly
to beam deviati0n, can cause noticeable ghost images as a result of 0ff-axis
internal reñecti0ns.
4) The clear aperture Of optical filters should not restrict the overall aperture
of the microscope. It is also Of critical importance that there is no leakage of
unfiltered light around the edge of the clear aperture.
5) Scratches and digs are measured in terms 0fmi1-spec standards; e.g., 80/
50 scratch/dig. The term digs includes such things as particles and small
bubbles imbedded inside a f11ter, and macroscopic inclusions in exposed coat-
ings.
6) Pinholes are small breaks in the coating of an interference filter, usually

caused by the presence of dust particles on the substrate during coating. Pin-
h01e size must be measured against standard maximum-tolerance pinholes
under specific conditions using a high-intensity illuminator.
Optical quality requirements for wide-field microscopes with K6hler

illumination
Most fluorescence microscopes are wide-jield epiñu0rescence systems that uti-
lize Kóhler illumination (illustrated in Figure 3, on page 6). Following is a
briefdescription:17
In Kóhler illumination, two adjustable diaphragms are provided for control
Of the light beam: the aperture diaphragm that gets imaged onto (i.e., is conju-
gate to) the back aperture of the objective, and thefield diaphragm that gets
imaged onto the in-f0cus specimen plane. The field diaphragm is positioned
near the lens that images the aperture diaphragm onto the objective back
aperture. The collector lens assembly focuses the arc lamp onto the aperture
15 This is strictly true only for light reflected at normal incidence. The value for light
reflected at non-normal incidence is modified by a cosine factor. For example, the
reflected wavefront distortion at 45 degrees angle of incidence is approximately 1.4
times the surface flatness.
16 For small angles of incidence, the angular deviation = (N- 1)a, where Nis the
refractive index of the glass in the filter, and a is the wedge angle. Most filters use
optical glass with a refractive index of approximately 1.5.
17 For an in-depth description of Kóhler illumination optics, please referto Inoué (1986),
or literature from the microscope manufacturers.

diaphragm and thus onto the objective back aperture. Three essential effects
are achieved with this configuration: 1) the image Of the arc lamp is com-
pletely out of focus at the specimen plane, thus creating a uniform field of
illumination; 2) the image of the field diaphragm appears in sharp focus on
the specimen, and it can be adjusted to preciser fill the field of view or isolate
any part of it; and 3) the numerical aperture of the illuminatng beam can be
adjusted (by adjusting the aperture diaphragm) without affecting the size of
the illuminated field.
With this in mind, some observations can be made regarding optical quality
requirements for this type Of microscope:
1) The illumination 0ptics of the microscope are designed for uniform illu-
mination of the specimen with minimal Hare, and not necessarily for faithfu1
imaging of the light source or apertures. Therefore excitation filters do not
require transmitted wavefr0nt dist0rtion Of imaging quality,18 but only what
is generally considered the industry standard Of optical quality. Likewise, the
surface ñatness of the dichroic mirror does not require imaging quality.
2) Variati0ns in the alignment Of the beamsplitter inside the f11ter cube assem-
bly will produce noticeable shifts in the position Of the illumination spot (i.e.,
the image of the field diaphragm on the specimen plane) when switching
filter cubes. For typical applications, an alignment Of 45 degrees plus or mi-
nus three arc-minutes is considered adequate. (Because the angle of the re-
ñected beam is always equal to the angle of the incident beam, the overall
angular deviation caused by this alignment tolerance would be twice this, i.e.,
plus or minus six arc-minutes.) When filter cubes are supplied with the
beamsplitter premounted, it is the responsibility of the supplier to properly
align the beamsplitter. In addition, care must be taken to assure proper align-
ment when the filter cube is installed into the microscope.
In contrast, variations in alignment have little effect on the apparant position
of the image in the field Of view, known as registration shift.
3) Similarly, the effect of angular deviati0n induced by wedge in the excita-
tion filter will be a shift in the position Of the illumination spot, but there will
be no registration shifts Of the image itself. Wedge must be controlled only to
within alignment tolerances Of the dichroic mirror mounted in the f11ter block
assembly.
4) Filters in the image path, including the dichroic beamsplitter as well as the
emission filter, generally do require transmitted wavefront dist0rtion of imag-
ing quality.
5) The effect of wedge in the emission filter and beamsplitter will be a regis-
tration shift of the image when switching filters or cubes. Wedge must be
controlled in these components.
In addition, variations in the thickness of the beamsplitter might cause regis-
tration shifts. The extent of this thickness-related shift depends on the micro-
scope: epiñuorescence microscopes wtih standard tube length 0ptics have a
light beam in the imaging path that is not perfectly collimated.19 They are
18 The term imaging quality denotes a level of optical quality that will not degrade the
overall performance of a specific optical system. For a typical microscope, a TWD of 1
wave per inch is generally considered adequate for optical filters and beamsplitters.
19 Most modern microscopes of this type have relay Ienses in the filter-block housing
that improve the collimation inside the cube. The sensitivity is reduced, but not
eliminated entirely.

therefore more sensitive than microscopes with injinity corrected 0ptics that
have, in principle, collimated light in the image path.
6) Since the excitation filter is usually positioned near the field diaphragm (a
point conjugate to the image plane in Kóhler illumination), pinholes in the
excitation filter are very noticeable and must be eliminated.
7) Autoñuorescence Of the dichroic beamsplitter must be minimized because
this is the one element that is both fully illuminated by excitation light and
also part Of the optical path of the image.
It is important to note that the beamsplitter requires separate and independent
specifications for surface ñatness and transmitted wavefront distorti0n.
Based on the above observations, some typical optical quality specifications
for f11ters used in wide-field epifluorescence microscopes are listed in Table 2.
TABLE 2
Typical optical quality specifications
for filters in a wide-field
epifluorescence microscope.
FI LTER TYPE
OPTICAL QUALITY
PARAMETER EXCITATION m BEAMSPLITTER
Surface flatness no speciñcation no specification <10 waves per inch

Transmitted wavefront no speciñcation 1 wave per inch 1 wave per inch
Wedge <6 arc minutes 1 arc minute 1 arc minute
Scratch/dig 80/50 60/40 40/40
Pinholes none minimal no specification*
Autoñuorescence no speciñcation moderate control minimal
*Measured as a dig using scratch/dig speciñcations.

When designing filters for use in confocal fluorescence microscopes FILTERS FOR
(CFMS), one must consider primarily the optical configuration of the CONFOCAL
microscope. Almost every make and model of CFM has a unique configura-
tion and therefore a unique set of optical specifications, but most systems can MICROSCOPY
be grouped into one of two basic categories: CFMS that utilize a Nipk0w-disk
mechanism for scanning the specimen, and those that scan with a laser beam.
The 0ptics used in each of these scanning methods are briefly described be-
low, by comparing them to the 0ptics of a standard wide-field microscope.
For a more detailed analysis Of CFM optics, such as depth of field, resolution,
and image generation techniques, please refer to the literature available from
the CFM manufacturers, as well as articles published in scientific journals
associated with microscopy 0r1aboratorytechniques for biology and genet-
ies.
OPTICAL QUALITY REQUIREMENTS FOR CONFOCAL

FLOURESCENCE MICROSCOPES
Nipkow-Disk Scanning CFMS
Figure 26 is a schematic illustration Of a CFM with broad- ?
band arc 1amp illumination, a Nipkow-disk scanning .
mechanism, and an image detector, either a camera or di- +
rect visual observation.
This microscope has an optical path similar in principle ºu .
to standard wide-field scopes (illustrated in Figure 3). The I%I Z:Sf ag 3£Íá?£f; ¡ ?
disk is set in a position conjugate to the specimen plane 5.'¿Í'ÍÍÍZ IV D U 0 *%
and must be uniformly illuminated over the entire field IÁ_J , , ' '
of view, usually with Kóh1er illumination. Therefore the 1 Relay º
transmitted wavefront requirements for the excitation fil- 1 , A'º ¡
ter, and the surface flatness requiremth for the dichroic 1 , p ,k
beamsplitter, are the same as for the wide-field microscope. ¡ Re 0 x
Similarly, the transmitted wavefront requirements for the 1
emission filter and beamsplitter are the same as for the _ _
wide-field microscope. The wedge requirements for the ¡ Ob'eºwe
various filters are, in general, also unchanged, although Specimen
variati0ns in the focal length of specific CFM designs FIGURE 26
might necessitate modifications Of these specifications. Schematic ºf a confocal
fluorescence microscope with
Laser Scanning CFMS Nipkow-disk scanning.
Figure 27 (page 26) is a schematic illustration of a CFM with laser illumina-
tion, a moving-mirror scanning mechanism, and photometric detection such
as a photomultiplier tube (PMT). The optical paths in this microscope design
are significantly different from those in wide-field microscopes. The two ma-
jor differences are
1) the pinhole (or slit) in the illuminator is imaged onto the specimen plane
via critical illumination, with the laser spot being directly focused onto the
sample; and
2) the image is formed by synchronously matching the train Of electrical signals
generated by the detector to the position Of the laser beam on the specimen,
similar to the image-f0rming mechanism of a television set. The electrical signals
from the detector correspond to the variati0ns in the intensity of the fluorescence
signal that passes through the conjugate pinhole (or slit) aperture.

In this configuration, the laser beam must remain undist0rted in order to
achieve the best focus at the specimen plane, and there must be minimal an-
gular deviati0n from the true optical path. In addition, most CFMS of this
type have relativer long optical path lengths that can magnify any angular
[| deviati0ns introduced by a filter. Theref0re the excitation filters and
Detectºrs/ " ) Emiss¡ 1 ers beamsplitters require a degree of optical quality and registration equal to or
7ººº'ºº III ' greater than that required for emission filters and
I 1 ,S;ºeºrggígnbºa st)'mºf g?$ilíalletsiltselirtss) beamsplitters. Most systems have transmitted wavefront dis-
|í|l = íE| , , torti0n and surface ñatness specifications on the order of
' * /' [| '. one wave per inch for excitation filters and beamsplitters.
. . .' ] The wedge specifications are similarly tighter, typically one
ZTÍÍ£Q$ZÍ¡'$0 Ii]. / [| arc-minute or less, in order to minimize alignment adjust-
|__ X ments when switching filters.
Exº º º " º'º In the emission optics, any components that are located be-
,S;º¿giggºgºa 5º'mºf tween the objective and the detection aperture must be of
¡ Objective imaging quality. In the example illustrated, this includes all
: of the emission filters and beamsplitters shown. Some systems might use a
W single aperture that would be located between the main excitation/emission
FIGURE 27 beamsplitter and the detection unit. In this case, the optical requirements for
Schematic ºf a laser scanning the filters within the detection unit might be less critical.
confocal fluorescence microscope
SPECTRAL REQUIREMENTS FOR CONFOCAL FLUORESCENCE

MICROSCOPES
Any unique spectral requirements generally stem from the type of illumina-
tion and detection in the microscope. Since the illumination and detection
systems Of Nipk0w-disk scanning CFMS differ from those 0f1aser-scanning
CFMS, most can again be grouped into one of these two categories.
Nipkow-Disk Scanning CFMS

Since the light source and the detection system of this configuration (de-
scribed above and illustrated in Figure 26) are the same as for standard wide-
f1eld EFMS, the spectral requirements are basically the same, with one exception:
because 0freduced signal intensity and the addition Of extra optical elements,
each of which produce undesirable reñecti0ns and scattering, excitation and
emission filter pairs must have very low cross-talk.20 In addition, extra care is
required in the quality control of pinholes in the filters.
Laser Scanning CFMS

Filters and beamsplitters for this configuration (described above and illus-
trated in Figure 27) are designed to work 0ptimally for the specific laser being
used. The most common laser is the arg0n-i0n laser, illustrated in Figure 22,
but other popular 1asers include helium-ne0n (543 or 633 nm output) and
argon-krypt0n (primarily 351-363, 488, 515, 568, and 647 nm output.) When
designng filters for use with lasers, the following considerations must be made:
1) Excitati0n filters must have a blocking range that covers the entire output
spectrum of the laser. Filters that block to 800 nm will be suitable for any of
the laser sources meti0ned above. One special considerati0n regarding the
filtering 0f1aser excitation is that excitation filters for lasers should attenuate
20 Some systems utilize polarized optics to reduce the effect of these reflections.

as much as possible by reflection in order to reduce the thermal load on the
filter. For example, UV excitation filters for arg0n-ion lasers should have high
reñectivity in the blue-green region Of the visible spectrum where most of the
laser power is concentrated.
2) The 1asers used in most CFMS are polarized, so knowledge of the polariza-
ti0n conditions is very useful when designng beamsplitters for 0ptimum per-
formance. The dichroic beamsplitter illustrated in Figure 10, for example,
was optimized for use in S-plane 0rientati0n with the 488 nm arg0n-i0n laser
line, enabling the beamsplitter to have greater throughput in, for example, a
band of fluorescent emission that cuts on at 500 nm. Note that fluorescent
emissi0ns are larger unpolarized even when the excitation light is polarized,
so the beamsplitter must efficiently transmit the fluorescence in both planes
Of polarizati0n. Knowledge Of the polarization conditions is especially useful
when designng multiple-bandpolychr0ic beamsplitters, which must have very
narr0w reñecti0n bands in order to maximize the transmission of the emis-
sion signals.
3) Emissi0n f11ters must have a blocking range on the short wavelength side Of
the band that includes all laser lines that might be used with the particular
filter. Blocking on the long wavelength side is recommended for applications
that use multiple probes, to improve selectivity between the probe signals.
Here the range of blocking only needs to cover the range Of the detector,
which can be as short as 700 nm for some PMTS (Figure 23.)

TABLE 3
Methods for applications using
multiple probes.
METHOD COMPON ENTS ADVANTAGES DISADVANTAG ES
1. Separate single-band filter Standard microscope. Nº extra equ¡pment 'S necessary. 3¿11|S,|i|£teous ¡magmg 'S not
sets. Brlghtest Images.
Imprecise imaging of combined
images.
-_ - . Simultaneous visual observation Reduced brightness.

2' MU band f||ter sets. Standard mmroscope. with no registration errors. .
Not recommended w¡th xenon arc
No extra equipment is required illumination.
th th ' | f'lt t.
(0 er an spec¡a ' er se ) Color balance is fixed.
3 . M5 It'-'¡ 5r?2¡33&% 223?ng Microscope with filter wheel or Seque_ntial yisual observation with EXtra equipment (a filter wheel or
slider in the illumination path. no reg¡strat¡on errors. shder) ¡s necessary.
Photo camera, or electronic _Precise registration of combined
camera with image processing. Images, and ad1ustable color
balance.
Optimized exciters offer brighter
image than method 2.
4. Multi-band beamsplitter Microscope with filter wheels or Exciters and emittere can be Extre equipment (two filterwheels
single-band emitters ahd sliders in both the illumination path d_es_¡|gnetd to tttrevg $rlghtness or sl¡ders) ¡s necessary.
exciters, Single camera. and ¡magmg path. s¡m| ar º me 0 ' Registration errors between
Photo camera, or electronic Reducti0n in registration error (by emitters might still be present.
camera with image processing. ehmmatmg movement of the
beamsplitter).
5. Multi-band beamsplitter Microscope with filter wheel or In addition to advantages in More pompli0a_ted apparatus_
single-band emitters ahd slider in the illumination path, method 4: requmng add¡t¡onal beamsphtters
eXC¡ÍGFSY multiple cameras. Beamsplitter assembly for Registration errors from emitters and cameras.
separating channels, each channel can be eliminated.
having a separate emission filter.
Additional applications are
supported (e.g., ratio imaging).
. - Any valid combination of exciter Brightness can be reduced by as

6' N%ñiih%%%ngígggglirt(tegr|?r$mg and emitter can be used. much as 80%, so special light
methods 4 and 5 sources are recommended (e.g.,
' laserillumination).

Y ¡ ] hen making a multiple-exposure photograph or multiple electronic im FI LTERS FOR
ages of specimens stained with several ñu0r0chromes, using separate
filter cubes in a standard microscope, there will be unav0idable registration MU LTIPLE PROBE
shifts between exposures. Varyng amounts Of wedge in the emission f11ter and APPLICATIONS
beamsplitter, variations in thickness and alignment of the beamsplitter, and
mechanical vibrati0n that 0ccurs when the cubes are switched, all contribute
to this shift. Although for some applications these effects can be reduced to
acceptable levels, many others require more sophisticated filter designs and
optical apparatus.
Listed in Table 3 are some available methods for applications involving mul-
tiple probes. These include the multi-band filter sets introduced earlier, illus-
trated in Figure 28, and various configurati0ns that utilize both multi-band
and single-band filter components. All Of these methods are designed to elimi-
nate the above-menti0ned registration errors. Method 2 offers the ability to
visually observe up to three colors simultaneously. (Methods 3 through 6 can
also do this ifmu1ti-band filters are added to the filter sets.) It should be noted
that this list is only a guide, not an exhaustive list of all possible methods and
configurations.
FIG URE 28
Spectra of a tripIe-band filter set designed for the dyes DAPI,
FITC, and Texas Red©- (Chroma Technology Corp. P/N 61002)
¡a
F|UOI'9 cence
_]
¿|. Excitation light
a ¿º,.t¿3
P | h '
¡¡ T ' I -b d
emission filter
1ºº
%T""""1"1 f'
" ""j"'B|0e'j " 'G'reén' f"j"j'""'jhéd'j"'j'

,,,Ex ,,,,,, , ,,E,m- ,,,E,X-, ,,Em,-, ,,,E,X-,, , , , , , , , ,,,Em-,,, , ,,
0 u 1 1 11 1 1 1 1
400 500 600 700
Wavelength (nm)

Abramowitz, M. (1993) Fluorescence Microscopy: The Essentials. Olympus America, Inc.
Brimrose Corp., Baltimore, MD. AOTF Spectroscopy (March, 1993 catalogue.)
Haugland, R.P. (1992) Handbook of Flu0rescenct Probes and Research Chemicals, 5th ed. Molecular Probes,
Eugene, OR.
Inoué, S. (1986) Video Microscopy. Plenum Press, New York.
Kasten, F. H. (1989) The 0rigins Of modern fluorescence microsc0py and fluorescent probes. In: Cell Structure and
Function by Microspectrophotometry (E. K0hen and J. G. Hirschberg, eds.). Academic Press, San Diego, CA.
Lakowicz, .]. (1983) Principles ofFluorescence Spectroscopy. Plenum Press, NewYork.
Schott Glasswerke, Mainz, Germany. Optical Glass Filters (catalogue)
References for Liquid Crystal Tunable Filters (p.16)
Cambridge Research and Instrumentation, Inc., Cambridge Ma. Varispec Tunable Imaging Filter (catalogue)
Morris, Hannah R., Clifford C. Hoyt, Peter Miller, and Patrik J. Tread0 (1996) Liquid crystal tunable filter Raman
chemical imaging. Applied Spectroscopy 502806-808
Hoyt, Clifford (1996) Liquid crystal tunable filters clear the way for imaging multipr0be fluorescence. Biophotonics
International July/August.

Acousto-optical tunable filter (AOTF) (p. 15) GLOSSARY
An active crystal device that works by setting up
radio-frequency acoustical vibrati0ns in the crystal (WITH PAGE REFERENCES)
and creating, in effect, a bulk transmission diffrac-
tion grating. By varying the frequency, one can rap-
idly tune the filter to diffract out a desired wavelength
of light and transmit this wavelength out of the de-
vice.
Angle of incidence (p. 12; fig. 9)
The angle between the optical axis of the light inci- Bandpass (P- 11; "9 6)
dent on the surface of a filter and the axis normal to An optical filter that has a well-defined short wave-
this surface. length cut-0n and long wavelength cut-0ff. Bandpass
filters are denoted by their center wavelength and
Angular deviation (p. 22) bandwidth.
A shift in the direction of light beam from the true
optical axis of the system, measured in units Of angle Bandwidth (P- 11; 9 6)
such as arcminutes (1/60 of a degree) or arcsec0nds Also FWHM (Fu11 Width at half ºf maximum trans-
(1/60 Of an arcminute). mission). For optical bandpass filters, typically the
separation between the cut-0n and cut-0ff wavelengths
Aperture diaphragm (p. 22) at 50% 0fpeak transmission. Sometimes a bandwidth
An adjustable diaphragm located in the illumination at, for example, 10% of peak transmission is speci-
0ptics, which controls the numerical aperture of the f1ed.
illuminating beam and affects the brightness Of the
beam. Barrier filter
See Emission filter.
Attenuation level (p. 11; fig. 6)
Also Blocking level. A measure of the 0ut-0f-band Blººk¡ 9 level
attenuati0n Of an optical filter, over an extended range Sºº Attenuation level.
of the spectrum. The attenuati0n level is often de- Blocking range (p. 11; fig. 6)
fined in units ºf optical dCHSÍÍY (see). The range of wavelengths over which an optical f11ter
Autofluorescence (p. 3) maintains a specified attenuati0n level.
In fluorescence microscopy, any fluorescence from Blocker (p. 14; fig. 15)
SUbSÍ3HCCS other than ñuorochromes, including pri- A thin-film interference coating that is incorporated
mary fluorescence from the specimen, fluorescence into a bandpass interference f11ter to extend the block-
from immersi0n oils and other media, and ñuores- ing range of the primary coating in the ñlter. A blocker
cence from glass optical components within the mi- is usually a very wide-band bandpass filter having
crosc0pe. high transmission in the band of the primary filter.
Average transmission (fig. 6) Brightf¡e|d (p. 3)
The average calculated ºver the useful transmission A kind of diascopic illumination in which the field of
region ºf a filter, rather than 0Vºf the entire spec- view is illuminated directly. Also, the type of con-
trum. For a bandpass filter, this region spans the denserused f01'th18kind0f1uuminat10n.
FWHM of the transmission band.
Center wavelength (CWL) (p. 11; fig. 6)
Background (P- 6) For optical bandpass filters, the arithmetic mean of
Any detectable light that is not a dCSÍYCd primary ºf the cut-0n and cut-0ff wavelengths at 50% of peak
indirect fluorescent emission. Sources Of background transmission.
include cross-talk between excitation and emission
filters, light leaking through pinholes in filters, and Clear aperture (p. 22) _ _ _ _
electronic noise in cameras, as well as The surface area of an 0pt1cal f11ter wh1ch15 free of
autoñuorescence. any defects or obstruct10ns. On 1nterference f11ters
the clear aperture is often delimited by an annulus of
metal or 0paque material.
BRATTLEBORO VERMONT 05301 USA G-1

Critical illumination (p. 25) Excitation and emission (p. 3)
A type of illumination optics in which the image of See Fluorescence.
the 11ght source 15f0c0sed0ntothe spe01men plane, Excitation filter (p. 6; fig. 4)
m contrast to Kohler 111ummat10n 0pt1cs. See also . . .
K hl '11 . ti Also Exc1ter. A color f11ter that transm1ts only those
º º mm º ' wavelengths ofthe illumination light that efficiently
Cross-talk (p. 12; fig. 7) excites a specific dye. See Emissi0n filter.
The minimum attenuati0n level (over a specified .
. . Exc¡ter
wavelength range) of two f11ters placed together 1n . .
. . . . See Exc1tat10n f11ter.
ser1es. The transm15510n spectrum of the beamsphtter
is sometimes included in this evaluation. Extinction coefficient (p. 5)
Darkfield (p. 8) Í,Q,ÍÍÍ$ Of the absorption characteristics Of a ñu0-
A kind of diasc0pic illumination in which the spec- '
imen is illuminated 0h1iquely, i.e., at angles that will Fading
not enter the obj ective directly. Also, the type of c0n- See Photobleaching.
denser used for this kind of illumination. Field diaphragm (p. 22)
Diascopic illumination (p. 8) An adjustable diaphragm located in the illumination
Illumination using light transmitth through the speci- optics, which controls the area 0fi11umination on the
men, using a condenser to focus the light. specimen.
Dichroic beamsplitter (pp. 6, 7; 7fn; fig. 4) Filter block
Also Dichroic mirror, Dichr0matic beamsplitter. A See Filter cube.
specíial 13irror Eousec1 ini the1 'fi111teí' 1cubedtthat 1Íe1ec- Filter cube (p. 6; fig. 4)
t1ye y re ects t _e exc1tatton lg t 1 tere y t e e)_(' A removable cube-shaped unit that holds a matched
c1ter andtransm1ts the ermtted ñeorescence. D1chr01c set of fluorescence filters. This set usually includes
beamsphtters can alsottl)e fºll,mg m any other par;0f a an excitation filter and emission filter but always in-
m1cr0-sc0pe system w ere lg t needs to be sp 1t or c1udes a dichroic beamsplitter.
combmed by wavelength.
E f'| 11 Filter glass (p. 13; 13fn)
Adge h| ter (p f ) , h h 1 _ Also Ahsorpti0n glass. Colored glass that is manu-
not er term or e1t er a S ortpass or 011gpass 0pt1- factured for technical and scientific applications. The
cal f11ter. The term usually denotes a f11ter w1th a most common types of filter glass use(1 in fluores-
very sharp cut-0n or cut-0ff. cence microscopy are: UV-transmitting black glass
Emission filter (p. 6, 7; fig. 4) filters; IR-absorbing heat filters; and yellow, orange,
Also Barrier filter, Emitter. A color filter that attenu- and red sharp-cut longpass filters. Filter glass selec-
ates all of the light transmitted by the excitation filter tively attenuates light by absorption, SO the spectral
and very efficiently transmits any fluorescence emit- performance is dependent on the thickness of the
ted by the specimen. glass.
Emitter Fluorescence (p. 3; 3fn)

See Emission filter. A molecular phenomenon in which a substance ab-
. . sorbs light, then radiates part Of this absorbed energy
Ep|fluorescence microscope (p. 6) .
. . . . as hght of another color, one of lower energy and
A fluorescence nucrosc0pe that111ummates the spec1- . .
. . . . . thus longer wavelength. Th1$ process IS known as
men ep15c0p1cally (1.e., w1th 11ght reflected onto the . . . . . . . .
. ) exc1tat10n and ermsswn. Fluorescence15 d15tmgmsh-
spec1men ' able from other types of luminescence in that the
Episcopic illumination (p. 6) process of excitation and emission 0ccurs nearly in-
Also Incident lightillunúnation. Illumination with light stantaneously (i.e., on the order of nanosec0nds.)
reflected onto the specimen rather than transmitted
thr hth . Th '11 . ti li ht' 11 t d Fluorescent probe (p. 3)
0ug e spec1rhen. e1 umma ng g 1.8 re ec e Also Fluorophore. A ñuor0chr0me that has been
through the 0h]ect1ve by means Of a beamsphtter that15 . . .
. . . . . con1ugated to an act1ve substance, such as a prote1n,
e1ther part1ally reñect1ve or a d1chr01c. . . . . . .
ant1body, or nucle1c ac1d, 1n order to select1vely sta1n
a targeted substance within the specimen.
G-2 CHROMA TECHNOLOGY CORP.

Fluorochrome (p. 3) age of the light source is completely out Of focus at
A fluorescent dye used either directly as a specimen the specimen plane.
Í1ta1n or c0í1]uggted to an act1ve substance to make a Longpass (LP) (p. 11; fig. 6)
uorescen pro e. An optical filter that attenuates shorter wavelengths
F | uo rop ho re and transmits longer wavelengths over the active range
See Fluorescent probe. Of the spectrum (which depends on the specific ap-
plication). LP filters are denoted by the cut-0n wave-
Front surface (p. 21) ) th t500/ f aktr . .
The side Of a beamsplitter that is designed to face the eng a º 0 pe ansrmssmn.
incident light. In a f11ter cube this is the side that faces Luminance
both the light source and the specimen. Beamsplitters See Radiance.
generally perform better when 0r1ented correctly. Nanometer (nm) (p. 4)
FWHM A unit of length commonly used for measurng the
See Bandwidth. wavelength Of light: 1 nm : 10 angstr0ms (A) = 10 9
HaIf-cone angle (p. 12) meters,. and 1000 nm _ 1 nucr0n
'
(p) _ 10 -6 meters.
The angle between the most 0h1ique ray of a conver- Near infrared (NIR) (p. 4; fig. 2)
gent or divergent light beam and the optical axis of The region of the electromagnetic spectrum ranging
the beam. See also Numerical aperture. in wavelength from approximately 750 to 2500 na-
Heat filter (p. 7) ºmºtºr5'
An optical filter that attenuates infrared radiati0n but NeutraI-density (ND) filter (p. 7)
transmits the visible. Attenuati0nis achieved by means An optical filter that attenuates light by an amount
of absorption (using filter glass), reñecti0n (using a independent Of the wavelength within the useful range
thin-film interference coating, often called a hot mir- Of the filter. Metal-c0ated f11ters typically have a wider
r0r), or a combination of the two. neutral range than glass filters and are more heat-
. tolerant.
Hot m¡rror
See Heat filter. Normal incidence (p. 12)
Indirect fluorescence (p. 3) An angle 0f1nc1dence of zero degrees.
Also Secondary fluorescence. In fluorescence mi- Numerical apetture (NA) (p. 8, 23)
crosc0py, fluorescence emitted by ñuor0chr0mes in- In the microscope, a measure of the effective cone-
tr0duced into a specimen as a stain or probe. See angle 0f1ight focused onto the specimen (NA of the
Primary fluorescence. condenser) 0r1ight captured by the obj ective (NA Of
Infinity-corrected optics (p. 24) the object1ve). A h1gh value 0fNA1mproves h0th the
. . . . br1ghtness and the resolut10n of the 1mage.
An 0pt1cal conf1gurat10n employed by some m1cr0- . . . .
. . . . . . (NA : N sm(0), where N IS the refract1ve 1ndex of
scopes, 1n wh1ch the object1ve forms an 1mage at1n- . . . .
. . . the med1um surroundmg the spec1men and 0 IS the
f1n1ty, and a secondary tube lens forms an 1mage at h 1f ) fth ). ht
the intermediateimage plane. (This intermediateim- a -c0ne ang eo e lg ')
age is focused in turn by the ocular.) This configura- Optical density (OD) (p. 11)
tion allows for a flexible distance between the objective A logarithmic unit of transmission: OD : -10g (T),
and ocular, because the distance between the 0hjec- where T is the transmission (0 £ T £ 1).
tive and the tube lens can be varied without affecting .
. . . . . Parallehsm
the 1mage-f0rrmng charactenst1cs Of the nucrosc0pe. See Wed e
Note that objectives designed for infinity-corrected g '
optics are not interchangeable with objectives designed Photobleaching (p. 5)
for standard tube-length optics. Also Fading. A ph0t0chemical reaction that causes
. the intensity of fluorescence to decrease with time.
Interference fl|ter
See Thin-ñlm interference coating. Pinholes (p. 22)
K6hler illumination (pp. 22_23; 22fn) Small breaks 1n the coat1ng of an1nterferenee f11ter,
. . . . . . usually caused by the presence of dust part1cles on
The type 0f111ummat10n 0pt1cs, usually used1n w1de- . .
. . . . . . the substrate durmg coat1ng.
f1e1d eplñuorescence nucroscopes, 1n wh1ch the 1m-

Polarization (p. 12; fig. 9) Registration shift (p. 23)
Restricti0n of the orientation of vibrati0n of the elec- A shift in the apparth position of the specimen that
tromagnetic waves in light. Unpolarized light consists 0ccurs when an optical element is inserted or removed,
of a mixture of transverse vibrati0ns orientated at all adjusted, switched with another element, etc.
angles perpend1cular to the d1rect10n Of propagat10n, Scratch/dig (p. 22)
and 0ccurr1ng at all phases relat1ve to each other. . . . . . .
. . . . A set of spec1f1cat10ns for def1n1ng the max1mum al-
When the v1brat10ns are restr1cted to one part1cular . .
. . . . . 10wable Slze and number Of scratches and d1gs on an
angle, the hght IS sa1d to be plane-polanzed. L1ght . .
. . 0pt1cal surface. The scratch/d1g values (e.g., 80/50)
can be part1ally as well as totally plane-polanzed . . . . . .
. . . . spec1fy the scratch w1dth 1n m1cr0ns and the d1g d1-
here polanzat10n IS the name Of a un1t of measure for . . .
. . . . ameter1ntens Of nucr0ns, respect1vely. Although ex-
quant1fymg the amount of polanzat10n. When the . . . . .
. . . . . ten51ve evaluat10n procedures ex1st 1f r1g0r0us
phase of the v1brat10ns var1es w1th angle 1n an 0r- . . . . . . .
. . . . . . standards must be ma1nta1ned (rmhtary spec1f1cat10n
dered fash10n, the 11ght IS sa1d to be elhpt1cally polar- . . .
. . . . . . . . . MIL-F-48616, for example), a quahtat1ve v15ual as-
1zed. (C1rcular polanzat10n IS a spec1al k1nd Of elhpt1cal . .
. . sessment of the scratches and d1gs usually suff1ces.
polanzat10n.)
. . fl .
When hght stnkes a specular surface at n0n-normal Secondery uorescence (p 3)
. . . . . See Ind1rect fluorescence.
1nc1dence, the component of the electr1c f1e1d v1bra-
ti0ns that is parallel to the plane of incidence of the Shortpass (SP) (p. 11; fig. 6)
surface (P-plane) behaves differently than the c0m- An optical filter that attenuates longer wavelengths
ponent that is perpendicular to the plane of incidence and transmits shorter wavelengths, over the active
(S-plane). This causes a polarizng effect in which range of the spectrum (which depends on the spe-
the orientati0n of the polarizati0n is aligned to the cific application). SP filters are denoted by the cut-
0rientati0n of the surface. Off wavelength at 50% of peak transmission.
Polychroic (p. 27) SignaI-to-noise (SIN) (p. 17)
A name for a dichroic beamsplitter that has multiple A measure of the brightness Of the desired ñuores-
reflection bands and transmission regions. cence (the signal) relative to the brightness of the
background (the noise).
P-plane
See Polarization. Slope (p. 12; fig. 8)
, A measure Of the sharpness Of the transition from the
anary fluorescence (p. 3) . . . . .
. . . transrmtt1ng reg10n to the block1ng reg10n of a color
In fluorescence m1crosc0py, fluorescent em15510ns
. . . . f11ter.
from the spec1men1tself, rather than em15510ns from
any fluor0chr0mes present in the specimen. See also Spectrofluorimeter (p. 3)
Autoñuorescence. An instrument used for measuring the excitation and
. . emission spectra of a fluorescent substance.
Quantum eff¡c¡ency (p. 5)
A measure of how efficient1y a ñuorochrome c0n- S-plane
verts absorbed radiati0n into emitted fluorescence. See Polarization.
Quenching (p. 5) Standard tube-Iength optics (p. 23)

Any chemical process which reduces the quantum An optical configuration employed by most micro-
efficiency of a fluor0chrome in situ. scopes, in which the objective forms an image at the
Radiance (p. 19) tntermed1ate1mage plane. (Th1$ 1ntermec11ate 1mage
. . . . IS focused 1n turn by the ocular.) The d15tance be-
A measure Of the rad10metnc br1ghtness of a hght . . .
. . . . tween the nosep1ece that holds the object1ve and the
source. Techmcally, the rad1ance IS the rad1ant flux .
. . . . . barre1 that holds the 0cu1ar15 f1xed at a standard length
em1tted per un1t sohd angle per un1t area of the hght . . .
. . . Of 160 mm, so that0b1ect1ves can be1nterchangeable
source. A common un1t IS watts per sterad1an per . . .
. . . between m1crosc0pes. See also Infm1ty-corrected.
square cent1meter. Lummance IS a measure of the
brightness of a light source as perceived by the hu- Stokes Shift (P- 4)
man eye (i.e., a photometric measure), commonly The shift in wavelength between the peak excitation
measuredin candelas per square centimeter. intensity and peak emission intensity of a fluoro-
chrome.
G-4 CHROMA TECHNOLOGY CORP.

Substrate (p. 7) Transmitted wavefront distortion (TWD)
The ground and polished piece of optical glass that is (p. 22; fig. 25)
used as a base for the beamsplitter coating. A measure of the distortion a plane wave of light
Surface flatness (p 21- fig 25) underg0es when transmitted through an optical ele-
A measure of the deviation of the surface of an 0pti- Ínentílmgaeltrbeld Í.n ÍtaCÍIOHÍIOI'SISHOU1t1pól3e3 ºf a wave-
ca1 element from a perfect plane, measured in frac- eng 0 VISI e lg (usua y or nm).
ti0ns or multiples of a wavelength of visible light Ultraviolet (UV) (p. 4; fig. 2)
(usually 550 or 630 nm). The region of the electromagnetic spectrum ranging
TE-mode in wavelength from approximately 100 to 400 na-
An0ther term for S-plane polarizati0n. (Short for gggnettíáóThreezdlst13%h/a1;ds arf910) ne;; OUV, trorg
transverse-electric mode.) See Polarization. tO nm, )m1 _ ' rom tO nm. an
3) vacuum-UV (VUV), below 190 nm. The terms
Thin-film interference coating (p. 14; fig. 13) UV-A and UV-B denote bands with distinct physi-
A type Of Optical coating composed 0fa stack micr0- 010gical effects: 320 to 380 nm and 280 to 320 nm
sc0pically thin layers of material. Although each ma- respectively.
ter1al ls-1ntrln51cally colorless, the reñect10ns created Wedge (p. 22; fig. 25)
ateach1nterface between the layers combme through . . .
. . Also Parallehsm. A measure of the dev1at10n of the
wave 1nterference to select1vely reflect some wave- t f f t" ) ) f f
lengths 0f1ight and transmit others. Thin-film inter- ou er SUI aces 0 an Op 1cai e ement rom per ect par-
ference coatings are the main component 0f allehsm,usually measuredmarcmmutes or arcsec0nds
interference filters, which consist Of one or more coat- Of angle.
ings separated by glass substrates, and frequently one Wide-field (p. 22)
or more layers Of filter glass. An epiñu0rescence microscope in which the full f1eld
TM-mo de of view is illuminated, in contrast to a confocal
Another term for P-plane polarization. (Short for epifluorescence microscope. The term brightfield is
transverse-ma netic m0de ) See Polarization also used, but this might be confused with traditional
g ' ' diasc0pic hrightñeldillunúnation.

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DIRECCION DIVISIONAL DE PATENTES

SUBDIRECCION DIVISIONAL DE EXAMEN_DE FONDO DE PATENTES AREAS MECANICA,
ELECTRICA Y DE REGISTROS DE DISENOS INDUSTRIALES Y MODELOS DE UTILIDAD
COORDINACION DEPARTAMENTAL DE EXAMEN DE FONDO AREA ELECTRICA
Expediente de Patente MX!a/2017l008919
Asunto: 1er. Reguisito de Fondo LPI. '
' ' ' ' , 019.

; fi NOF |ÍO: &&RREO FAC?URA
Carmen Gabriela REYES FUCHS ' " ,

Centenario No. 56 , A
Del Carmen " _ ' Nº' ¿.0 1 , 0031
04100, COYOACAN, Ciudad de México, México Í º á 1fg ¿j ¿ ,
» _ DIRE8%I%RA 20WSIONAL
REF: Su solicitud No. MXla/201?M891?3%?a$nºepr£sggigdaeiá 3;¿¡ _ ¿_e . , j
Como resultado del exámen__dg fondo ?eá¡zadá %ºñ fandámentoenbsíahí;úlos 53 de ¡a Ley de la Propiedad
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en su escrito No. MX/EIZOW7/092%12Ídéí1&1mg$_dici%bge de_gb£fí gugfºda* f Spuesfá al oficio de requisitos No. 72923
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Figuras: 1, 2, 3, 4, como ong¡nalmeñíg?ggmn présentáda&,díbuyos12 3, 41bérñówfueron presentados en su escrito

No. MX/E/2017/092212 del 11 de dICIWWW& ZÓW &U& ©Wtijaal oficio de requisitos No. 72923 del 8 de
septiembre de 2017.
1.- Las figuras 1, 2, 3 y 4 presentadas en el escrito MXIEI2017I092212 del 11 de diciembre de 2017, contiene materia
adicional que da mayor alcance a lo contenido en la solicitud original considerada en su conjunto de la invención, ya
que aparece el siguiente título EJEMPLOS VISUALES PRUEBA PARA LA SOLICITUD DE PANTENTE BREVE
DESCRIPCIÓN DE LAS FIGURAS , además cada figura 1, 2 3 y 4 menciona características técnicas que no estaban
presentes en la solicitud de patente MX/a/2017/008919 como originalmente se presentaron ante el IMPI. Dichas
características no se encuentran contenida en la materia originalmente presentada en su conjunto de su solicitud de
MX/2019/8369
A; m'1al NC 550 ( o!. Pueblo de Santa María "Tepepan. Del. Xochimi|co, .l. >0220. (í¡t.uííaá í'í :' f-f1eé,» s< <;
(58) 53340700 www gob.mx/impi
f,? ) ;ív4, »'Xñf:» < 5??$ f Íá'3ºí ffííííi fºi??? ffí$a;r" %:fmsíiwna .f; axn¿aá;;
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patente; contraviniendo así lo establecido en el artículo 558is de Ia LPI, por lo que deberá indicar en qué parte de la
solicitud originalmente presentada se encuentran contenidas las características que ahí se mencionan La figura 1 es
una imagen que presenta tonalidades que solo observan del negro al blanco pasando por una escala de grises y
utiliza la técnicas de microscopía de campo claro, se observan restos humanos de indiviudo de 60 años, causa de
muerte: cancer metastático. Se utilizó un objetivo 20; está muestra sirve de ejemplo para comparar los resultados del
método motivo de la presente solicitud con anteriores metodos utilizados, denotando la diferencia que existe entre un
proceso y el otro. La imagen se tomó con una cámara Nikon D800 en formato RA W'. "La figura 2 es una imagen que
presenta fluorescencia utilizando las técnicas de microscopía de fluorescencia con tos determinados filtros descritos
en el proceso de dicha solicitud proveniente de restos humanos de indiviudo de 60 años, causa de muerte; cancer
metastático. Se utilizó un objetivo 40x, en está muestrq log golqres predominantes son los azules es decir que tiene un
paso de bagde¿ de 450 nm ¡? 500 nm casi no se eqeaptíaror1£ot¡bs 'ao/of?s a escepción de un raja de igual intensidad a
la que se'rhuestfrá aqÚí. 1% imagen se tomó con una cámara Nikon D800,enfongto RA WC La figura 3 es una imagen
que presenta f/uorescencia utilizan,_d0 *las técnicas de,;aúegggggopia de ñuore$ceacia con los determinados filtros
descntos en el prpceso de la p(ea enle'solicitud, pro %%&3?;%manos *dgzxí¿ indiviudo de 80 años, causa de
muerte; LeupemiaMíe/bide: $gá íiiilizó un ob¡$y&2358290 $ ¿e ¡eggsggpectio ¿'de color tiene un rango desde los
colores cálidos hasta los fríos, es deoigºde ¡03330Ma ¡&Ú'nW prétdes intensidades dependiendo la luz de
onda como se muestra aquí. La ¡m&gn sg gg¿g£gngágg W33W%p formato RA W'. La figura 4 es una
imagen donde se utilizo un filtro pol&yZádgá,ge ñeááaé ; _ gopñpúe$b&,er©8éd?nte de ree;os humanos de indiviudo
de de 80 años causa de muen*e; Muerte 7fla$)raf EslaM prg&cº g%f&f $ñ?3bfdekmicroscapio claridad u oscuridad,
según que los dos r,ícoles esth paraley¿fáy$*crz[iáéád£ WWW ÓWV%WSQÍQ blapo". redámadas en las figuras 1,
2, 3 y 4 presentadas é'nº'el esbiñó NaWE/20£lº 2% ¿ ' 11*d g3mefu9m dg 2917 o en su defecto realizar las
modificaciones corresgondientes á_la$%úotasyd ': faaéífí3 B gra supexgñb a¿ígfk% enciopado; o bien, eliminar la
materia adicional de las notas présen%agg sa&n hgáh_ pángo el*párrh$o.$5E/WP&Q$ VISUALES PRUEBA
PARA LA SOLICITUD DE_PANTENTE B_RE %% $$CRWCÍQQMWQUR&Sera sugg ¿r¡gg|o antes menc¡onado.
__/(3 ? ], WW =, , .ayy £ u 7 »a Z ¿
2.- Las objeciones que:,se exponen awm gmºniglatlvasa&a*&ar£ad dé Io_¿s_* :dfbujggs son con fundamento en el
artículo 30 del RLPI. . ' _, :_ 3 " " , _ ¡?!
2.1 Las figuras 2, 3 y 4, n oég,gmplen dºn 10 egñpylaéíd%íá ar3ng¡o 9 ffáéción || del Acuerdo de las Reglas para la
presentación de solicitudes ante eHMI3I. pogc'10eééfáq a oqta r.jf _ *
Por lo tanto, se deberán hacer los eati1Qíp s_¡ºñeóg8g£1oáºcewe£abjetíºgúefgygerá»: ,e$ta objeción.
, ,? ¿? :,,¿ " '
3. Las objeciones que se exponen a continuación relatí%a a fá claridad de las reivindicaciones, son con fundamento
en los artículos 47 fracción III de 13 LPI y 29 del RLPI.
Las reivindicaciones presentan problemas de claridad debido a:
3.1 La reivindicación independiente 1, reclama: un filtro () cubo de excitación o emisión que permite una excitación de
filtro
longitud de onda de 372 nm y una emisión de 456 nm dentro del denominado U que refiere a Ultravioletas; un
dicha
o cubo de excitación"; sin embargo, el término o" es indefinido y como tal no deja claro el alcance de
reivindicación.
una cámara fotográfiea o
3.2 La reivindicación dependiente 5, reclama: "preferentemente se documenta conectando
de ducha
de video, al microscopio."; sin embargo, el término o es indefinido y como tal no deja claro el alcance
MX/2019/8369 Pag. 2
:»,¿f 1 *º » ii??? 10%. $'Hv*fío zíw mew í*»*wsrí:é 5<*;.3<3;w=, 3<:_,n hsm Mv??? º,' =:=í w? <% ?*%¿f> i»:,l_=
'L'»º1 f: :;4v: r'£' ss$ www gob mxíimpí
, r; :: , sf wí %, Íií'v'ífííg? x;"???í Í'f;¿* ? Ezzííñ,ií;ífé º' xi; >r;fí ;
IMPI f
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reivindicación.
3.3 La reivindicación independiente 8, reclama: un medio fotográfico o de vídeo". Sin embargo, el término o" es
indefinido y como tal no deja claro el alcance de dicha reivindicación.
Por lo tanto, se deberán hacer los cambios necesarios con el objetivo de superar estas objeciones.
4.- En cumplimiento a lo dispuesto por el artículo 17 de Ia LPI el cual se establece que para determinar si una
invención es nueva y resultado de una actividad inventiva se considerara el estado de la técnica en la fecha de la
presentación de la solicitud de patente o en supasb, $e3aápriaric¿ad reconocida, se le comunica que al determinar el
estado de la técnica de acuerdo al artícqu 12 fr á*ecíón?ll y el árribá citgdgi, ge encontraron los siguientes documentos
relevantes: ' '
D1 HANDBOOK OF OPTICAL FILTE BS F9RFLUQRBSQENQEM£CRDSQOPY DE JAY REICHMAN DE JUNIO

DE 1998 bttgs://degts.washiagtonfggjf¿Ikeakíbá'gº'Eaók3&f ¿
; 1?" , ¡ww M ? , ,?g? ? w
02 CN 106080004 (A) _»TIRCOT mmm; MQQE;1 985 ¡ if»
httºs://worldwide.esºacéñet.com/gubi¡cat¡gjkt
?éñóg_gº¡ j_t%g,&&!á
3&ad|'acent3&e&ocgjgáep E?á_FT=D£'d&'=19 M Z % 71, X_K_Q_= Ajtg_
_, . Ív ¡ ? J ??? , W?? Wºm N º
Los documentos citados se puede,;1 cd&;í$úka¡euel%xgg &ntéºfxºtel a&érñá é? áé,_quuentran a» disposición pública en
la base de datos de _accesg gfatúftó *deºatáuca&ge£tíbourqgnteg _ debégé$t& s a tniyeP ¿mundial denominada
ESPACENET en |as ,gjíggccioue_g,9e irgt em% bp|¡ .m/f
httgs//deºls washingtdníggu/ke&º hgg mk3fº ;;Í.' N, 'ij » 5
¡ , ' , , % íw' , ' "? ,
Con base en el contenídd¡gie Ios d&gme?£o;º£añte9º&á£e gefsggeffag,kj& giguiemef _
4.1 La reivindicación indepeh¿q ifg¿jtg 1 ñb óúmp1eyoqugt reqyígjtééég; actividad ¡nventiv3ºestablecido en el artículo 16 de

Ia LPI por ser evidente derivadb deºl íág&adáíáe ia»técnif©a cbn íenido en lºe gocg mentoj_$ D1 y DZ:
Para la reivindicación 1, D1 divulgáf ; lñ¡grgggeg ggrg£ogmgugung wmagena color (página 3, primer párrafo),
caracterizado porque comprende los siguiañeg ,pág;gágzºf . º
¡. depositar una muestra sobre un medio de sopoñe para;£%pbb$érvado bajo microscopio (figuras 3, 4);
ii. realizar observaciones a través de dicho microscopio con el método de epifluorescencia (página 6 a la página 8 a
primer párrafo; figuras 3, 4), por reflexión y de fluorescencia con un filtro de dos bandas (página 6, último párrafo a
la página 8 a primer párrafo; página 21, primer párrafo; figuras 3, 4, 6, 24), con una longitud de onda del 490nm a!
Interference Blue Filter) y un filtro o (ver objeción 3.1) cubo de excitación o (ver objeción 3.1) emisión que permite una
excitación de longitud de onda de 372 nm y una emisión de 456 nm dentro del denominado U que refiere a
Ultravioletas (página 4, figura 2; primer párrafo página 6, último párrafo a la página 8 a primer párrafo); un filtro o
(ver objeción 3.1) cubo de excitación de banda angosta denominado U-MNB SP 470nm-490 nm, DM 500, BA 515
(figura 28; tabla 3).
MX/2019/8369 Pág. 3
¡ ?(s'fíí'áí N ; i2'3<;_ (, a.;é í%¡s. ?Zí>ifí;<ís;*55 11;3M 1rí;¡ '%'??¡,,>< f;; , %>fo LX1£.>?'1¡;¡3?¡£4'< 3»?;13,/í >, (._¿s,w,í¿ 535» W z;
, íg»% ; ' >f: :;4>1': fa,'sí.r www g0i3.mx¡impí
. >'í ¿ _% _mí2?_? <'í»;ºi *" ºzºjf: ?H ? Í,'> .,f: ;1¿ ? ; '£ !
] M P 1 && *º* ?,
» .-, r:3? &; ms,
1% 3'W
Sin embargo, el D1 no menciona de materiales incinerados, de material incinerado; pero el DZ si lo hacé en el

primer párrafo.
Por lo tanto, es evidente para un técnico en la materia deducir el proceso reivindicado a partir del estado de la técnica
mencionado en los documentos D1 y 02.
En consecuencia, la reivindicación independiente 1 (así como sus dependientes) carece de actividad inventiva
De acuerdo con lo dispuesto en los artículos 55 de,,la L9,l y 45 del RLPI, deberá realizar las aclaraciones necesarias
sobre la actividad inventiva de Ia invenciQn 'reñííggigad,a Íeipaétóí a Ia$ r ofe_rencias citadas señalando las diferencias
entre ambos, y en su caso hacer las modificaciones correspondientes íakf'gcgmítulp reivindicatorio, para cumplir así con
lo establecido en los ar1ículos 16 y 12 fracción III de Ia LPL _ , _,
4.2 La reivindicación indepenqié'ñté 8, no cumglé&cáñf& r%áiófdéfffºí$ºpdad estaf$lec"ido en el artículo 16 de la LPI

por estar anticipadas en el D2,_3i momtho dém strar? méncº'ríarz' % 1
. . . ., . _ ?I'i:?$º fí&aº , ' . . , . .
Para la re¡vmd¡cac¡on 1: Una ¡magéz,1__fo;ghgda dg,aáu£r% ágfo,ñég& hfl9en las ºrggymducac¡ones antenores,
caracterizada porque muestra un materiaí fr£jñeragfá,&gg,&of ;;me t¿ii%&m fa?q9;áí£co o de video (ver objeción 3.3, a
pesar de ello en el abstract, summary 05% en áef? unv3fxcíórñ , , , : _
_ » , ¿v » ",X ;, , ; Í) íºí,j¿gºg if ? , : M" %% ¿ 5
Por lo tanto, la reivindicación indepéñc%$$e 8 carbtu£?"hq . : *? % /
De acuerdo con lo digpwgsto ¿q [ps ádígul&$5£déi&WW%d $hdebef% $áilzar 1a;$_58alarac¡ones necesanas

sobre la novedad défº 3á'ººínveñ'chn reñm'íd & :fgé ñj9étqfáyajááWeférégf&á;,j&;g'dásíseñalañ'd9 las diferencias entre
ambos, y en su casó haber Ias m081f49530ges corf% bdndfégt& af»báfbítulo»&e?vin9icatoriq, para cumplir así con lo
establecudo en los articulos 16 y 1'gfg,f,_r_?cc¡ºM£,9 Ia% &l g ; ,;
Se anexan D1 y D2. : ; WW % ?
Las aclarac¡ones o mod¡f¡camaúggaíg%a¿uzadg&ya séa%en Wrip$iór;y%n 105 d1bujg$ __y/o en las reuvmd¡cacuones, no
deberán contener materia adicioriái__33'tí11á rfj ayog__,áigaíipe cíué,la máteria"ºprequ%qíí á'íiginalmente en la solicitud y/o
elementos que den soporte a reiWñ3igjáci&nes*ºádjóióñºaies, dwiál mane,raq11e se cumpla con lo establecido en el
artículo 55 BIS LPI. , j, ,,
Asimismo. deberá efectuar el pago que establece la tarifá ví©en'fe y exhibir el comprobante correspondiente.
Para que satisfaga estos requisitos, se le concede un plazo de dos meses contados a partir del día siguiente a la fecha
en que se le notifique el presente oficio, apercibido que de no cumplir este requerimiento en el plazo señalado se
considerará abandonada su solicitud de patente de conformidad con lo dispuesto por los arts. 55 y 58 de la Ley de la
Propiedad Industrial.
El presente oficio corresponde al primer requerimiento de examen de fondo, el cual se emite, además de lo
fundamentado anteriormente, conforme al artículo 13 del Acuerdo por el que se establecen Reglas y Criterios para la
resolución de diversos trámites ante el Instituto Mexicano de la Propiedad industrial, publicado en el Diario Oficial de la
Federación el día 9 de agosto de 2004.
MX/2019/8369 ' Pág. 4

; :?ír.» ' ' ; <) % )! ??'? *E.>i '>fj=x %; mga M:;ríz'; 'E" ' gºwp. , 3"> ->í X?>£?a zv m £ ' M Q ¿,f:s?;eé e í &? fx?<<?xív.z z
(Faía; 33 ¿, /,,, » www.gohmx/¡mpé
; < <f ,.t ¿ < $< *á i í s:féiáí<í> <15< -<5¿ :,»<, ;í > ;';séfeam; ¿:Í 5.; ;'?
1 M P 1 fyv r <
4 _ , . . '
% <
43>¡S'l í _l'1jfi'£3 f;>1w1,_Pí/ií.;¿íyi*dl,r
El suscrito firma el presente oficio con fundamento en los artículos 6º fracciones III y XI y 7º bis 2 de la Ley de la
Propiedad Industrial (Diario Oficial de la Federación (D.O.F.) 27/06/1991, reformada el 02/08/1994, 25/10/1996
26/12/1997, 17/05/1999, 26/01/2004, 16/06/2005, 25/01/2006, 06/05/2009, 06/01/2010, 18/06/2010, 28/06/2010,
27/01/2012 y 09/04/2012); artículos 1º, 3º fracción V inciso 3) sub inciso iii) segundo guión, 4º y 12º fracciones I, II, III,
IV y VI del Reglamento del Instituto Mexicano de la Propiedad Industrial (D.O.F. 14/12/1999, reformado el 01/07/2002,
15/07/2004, 28/07/2004 y 7/09/2007); artículos 1º, 3º, 5º fracción V inciso 3) sub inciso iii) segundo guión, 16
fracciones |, II, III, IV y VI y 30 del Estatuto Orgánico del Instituto Mexicano de la Propiedad Industrial (D.O.F.
27/12/1999, reformado el 10/10/2002, 29/07/2004, 04/08/2004 y 13/09/2007); 1º, 3º y 5º incisos 0), e), g) e i) y
penúltimo párrafo del Acuerdo que delega facultadgs,en,£lqs Qirgctores Generales Adjuntos, Coordinador, Directores
Divisionales, Titulares de las Oficinas Regional es,_¿Sgbfglire&orás Divisiºnales, Coordinadores Departamentales y otros
subalternos del Instituto Mexicano de ia Prºpiedad Industrial. (Dí(3º.Ff 15112/1999, reformado el 04/02/2000,
29/07/2004, 04/08/2004 y 13/09/2007): '
,, ¿ *º*<'f , , <
As¡m¡smo, el presente docunmgnto electron¡coyhgºghdº8f 'rgeQ¡axgéíg%ggo de Ia f|rma electron¡ca avanzada por el

servidor público competente,,afnparaci? porunse$gfsgádd,d ggv&n& Qlágíecha de gu elaboración, y es válido de
conformidad con lo dispuesto en Io&%úmulggp£y%&h£c$á% <<lg wagní?í&,í%afág_lectrón¡ca,¿Avanzada y artículo 12 de
su Reglamento; y 1º fraCción III; 2ºíracgn;an2Q$wcngÍ,Ó633 3261 ERdQAcuen/opor el que se establecen
lineamientos en materia,de servicios elecfor%éog dá,ln£3;tuta M e>¿¡tá nb d e,láñrópbúad Industrial, en los trámites que
se indican. , ¿Ú ' Í " '
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COORDINADORA DEPARTAM,ENTALDEEXAM;N ( , / ¿' , , ¿
DE FONDO AREA EE,E&RICAÍ , . º " , _' » '
MARINA OLIMPIA C¿A'STFRO ALVEAR í
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E -_ " .!'=-i.-u I¡=': ". E Caáena Orígin,aj v,, $ ,»4, ¿ , ¿,

_ ' : _ " . :- %BiNA OBÍMPIA QQÓSJR%AI¿X£;ARÍUQQ£4000000%0891202815g;y¡ci0 de Administración
:" . -I":q' 'B&% ;300|E M>62819_36W017%003949|W5HRéqúis"gsó qefondo LPI]1677|'1'SAIPág/s)
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__:T.'. _."|!"'1l_._.':1-: ¡' YCMOijW+30482+XyiQC7DW¿/ÍWW3Fka4um1UyyHF(ieVpk(ic5m7N2thzl,I+aSI1SI1eÓZI4MIYM
- | '- -¡¡= 1"-F'nl:l' ZhZNUÍípUBLvQSSr3d21rrUlX(iaqc8ij53nPV9LYnUQ <S7NCWE2WV7ZZIUlUlvunYth9y9kSzrc3N
E . . ,' --.- _ _D_1'.I:-._. ' /Ltwhg9(jyz+3ClhdeYr0A==
Para verificar la autenticidad del presente documento, podrá ingresar a la página electrónica https://vulidadocumcnto.impí.goh.mx/. escaneandn el código
QR que aparece a un costado de la FIEL (Firma Electrónica Avanzada) del Servidor Público que signó el mismo. indicando. en su caso. el tipo de
documento que pretende validar(solicitud, acuse, oficio o promoción); 10 anterior con fundamento en lo dispuesto por los artículos lº fracción . 2º
fracciones II y V. 25. 26 BIS y 26 TER del Acuerdo por el que se establecen lineamientos en materia de servicios electrónicos del instituto Mexicano de
la Propiedad Industrial, en los trámites que se indican; en caso de no contar con lector QR o en su defecto el Código no pueda ser leído por su dispositivo,
puede digitar en la página antes referida el siguiente Código: 80112thL12b/E8+inBDZlZ ngw=
MX/2019/8369 Pág_ 5
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<, í-é ºf; ¡» 35 < 4<.3 / f*<) wwwgeb.mx¡impí

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NAL DE PATENTES

17, contiene materia

las Reglas para la

. , . 'te una excitación de

ica avanzada por el

|.111X/, escaneando el código

Three-dimensional stereoscopic imaging process for human ashes

Summary of the invention

2 CHROMA TECHNOLOGY CORP.

EXCITATION AND EMISSION SPECTRA

0300 400 500 600 700 800 900 FIGURE 1

BRATTLEBORO VERMONT 05301 USA 3

NEAR uv v GREEN Y DARK NEAR IR >

On the short-wavelength end of the visible spectrum is the near ultraviolet

4 CHROMA TECHNOLOGY CORP.

BRIGHTNESS OF THE FLUORESCENCE SIGNAL

BRATTLEBORO VERMONT 05301 USA 5

Field Aperture Filter

. Collctor Arc Iamp

An epiñuorescence microscope using Oil immersi0n, but without any filters

TYPES OF FILTERS USED IN FLUORESCENCE MICROSCOPY

6 CHROMA TECHNOLOGY CORP.

UV or U Ultraviolet excitation for dyes such as DAPI and Beam59"ttef

3) The dichroic beamsplitter (also called the dichroic mirror or dichro

1) A heatfilter, also called a hot mirror, is incorporated into the illuminator

BRATTLEBORO VERMONT 05301 USA 7

THE EVOLUTION OF THE FLUORESCENCE MICROSCOPE4

(After Kasten, 1989.) -¿ "

8 CHROMA TECHNOLOGY CORP.

8 Assuming nonmetallic specimens.

BRATTLEBORO VERMONT 05301 USA 9

10 CHROMA TECHNOLOGY CORP.

Some of the important terms used to describe the spectral performance of

2) Longpass and shortpass cut-0n filters (LP and SP)

OD : log(T) or OD : log(%T / 100) B) (Bandpass)

10 Full Width at Half of Maximum Transmission

BRATTLEBORO VERMONT 05301 USA 11

12 CHROMA TECHNOLOGY CORP.

Colored F||ter Glass

Colored filter glass, also called absorption glass, is the _ _

" Some minor exceptions are 1) sharp-cut Iongpass filter FIGURE 12

BRATTLEBORO VERMONT 05301 USA 13

Iní|;hñnt 3223fá3d$º£1erference Included in the category of filter glass are polymer-

14 CHROMA TECHNOLOGY CORP.

Advantages of the AOTF include: CW ? "

1) the ability to change to any wavelength within microseconds, perform º' -% e

BRATTLEBORO VERMONT 05301 USA 15

Liquid Crystal Tunable Filters*

16 CHROMA TECHNOLOGY CORP.

BRATTLEBORO VERMONT 05301 USA 17

18 CHROMA TECHNOLOGY CORP.

BRATTLEBORO VERMONT 05301 USA 19

20 CHROMA TECHNOLOGY CORP.

Opt|cal q0ahty perameters Wedge angle ¡,

BRATTLEBORO VERMONT 05301 USA 21

6) Pinholes are small breaks in the coating of an interference filter, usually

Optical quality requirements for wide-field microscopes with K6hler

22 CHROMA TECHNOLOGY CORP.

BRATTLEBORO VERMONT 05301 USA 23

Surface flatness no speciñcation no specification <10 waves per inch

*Measured as a dig using scratch/dig speciñcations.

24 CHROMA TECHNOLOGY CORP.

OPTICAL QUALITY REQUIREMENTS FOR CONFOCAL