Procesamiento de Imágenes

para Biología y Medicina IMA INA

Centro de Obtención y Análisis de Imágenes Biomédicas

Adquisición y formación de imágenes

Microscopía óptica
 Microscopía óptica

• Detección y conteo de fotones provenientes de la muestra.

• Luz incidente y reflejada.

• Widefield, fluorescencia.

• Interpretamos “lo que fluoresce”.

• No son las características, estructuras o moléculas de interés.

• Bajo ciertas hipótesis generales (plausibles) esa interpretación está

de acuerdo a la “realidad”.

• Conocer los fenómenos que afectan la formación de la imagen.

• Determinantes en la cuantificación y determinación de su origen.

 Adquisición de una imagen

Cuantificación
de fotones y
128 almacenaje
CHAPTER 14. FROM PHOTONS TO PIXELS
Detección de
fotones

Fotones
detectados

Fotones no
detectados
Preparación
de la
(a)muestra
The basic setup (b) The specimen
Excitación del (c) Light is emitted; some
Emisión de
is illuminated; fluo- enters the objective lens and
fluoróforo
rophores become ex- fotones del
is detected
cited High-energy excited
fluoróforo
state levels

Figure 14.1: A (very) simplified diagram showing the steps of image formation in
fluorescence microscopy. Absorbed
Emitted
Energy absorbed

fluorescence
excitation
Energy loss

light
light

14.1.1 Recording images

hc
Once a specimen has been E =
prepared and is waiting underneath the microscope,
the basic process of recording a fluorescence image comprises four main steps
Ground state

Fluorescence
(summarized in Figure 14.1): lifetime (ns)
 Incertidumbre y errores
PSF: Point Spread Function
• Incertidumbre en el espacio (blur)

• Fotones de un punto no terminan en el mismo “pixel”.

!
!
!
• Errores en medidas de (co)localización, tamaño, …

• Incertidumbre en el intensidad (ruido)

• Emisión aleatoria, y ruido electrónico.

• Errores en las medidas de intensidad (SNR).

(a)
(a)
(a)
Ideal
Ideal
Ideal
imaging
imaging
imaging
––a–adirect
adirect
direct
view
view
view
ofofof
‘reality’
‘reality’
‘reality’

!
!
! !
!
!

imagen “ideal” (b)

(b)
(b)
More
More
More
realistic
realistic
realistic
imaging
imaging
imaging––(PSF)
+ blur a–ablurred
ablurred
blurred
and
and
and
noisy
noisy
noisy
view
view
view + blur + ruido
 Modelo de formación de la imagen
14.1. THE BIG PICTURE OF FLUORESCENCE IMAGING 129 14.1. THE BIG PICTURE OF FLUORESCENCE IMAGING 129

˜ y) = I(x, y) ⇤ h(x, y) + n(x, y)

I(x,
! !

(a) Ideal imaging – a direct view of ‘reality’ (a) Ideal imaging – a direct view of ‘reality’

The universal reflected light vertical illuminator is interposed between the ob-
servation viewing tubes and the nosepiece carrying the objectives. (Figs. I & K)

˜ y) ⇤=!h(x,
Fig. I

˜ y) =! I(x, ˜ y) != I(x, y) ⇤
Universal Reflected Light
Illuminator mounted on an

y) ⇤ h(x, y)
I(x,y)
upright microscope

I(x, The illuminator is designed to direct light onto the specimen by first passing the
!
I(x,
light through the microscope objective on the way toward the specimen and then
using that same objective to capture the light being emitted by the specimen.
(Fig. J)

Fig. J
Fluorescence Reflected
Light (Diagrammatic)

(b) More realistic imaging – a blurred and noisy view (b) More realistic imaging – a blurred and noisy view

Figure 14.2: The di↵erence between what we might wish to image, and what we actually Figure 14.2: The di↵erence between what we might wish to image, and what we actually
PAGE 9 / REFLECTED LIGHT FLUORESCENCE ILLUMINATOR

can image. In both cases (a and b), the ‘real’ scene is shown on the left. Ideally, the can image. In both cases (a and b), the ‘real’ scene is shown on the left. Ideally, the
small coloured spots in reality would directly map to coloured spots of a related size and small coloured spots in reality would directly map to coloured spots of a related size and
separation in the image (a). However, the light emitted from these spots would actually separation in the image (a). However, the light emitted from these spots would actually
end up producing larger objects in the image, which can then blur together (b, centre). end up producing larger objects in the image, which can then blur together (b, centre).
Noise is further added to this blurriness to give us the best image we can really record (b, Noise is further added to this blurriness to give us the best image we can really record (b,
right). right).

these quantifications pixel values are determined. A larger charge indicates these quantifications pixel values are determined. A larger charge indicates
more photons, which translates into a higher pixel value. more photons, which translates into a higher pixel value.
˜ y) = I(x, y) ⇤ h(x, y) + n(x, y)
I(x,
14.1.2 Errors and imprecisions 14.1.2 Errors and imprecisions
From the above summary, it is clear that we are quite some distance away from From the above summary, it is clear that we are quite some distance away from
knowing exactly how much light is emitted from the specimen: most photons do knowing exactly how much light is emitted from the specimen: most photons do
not reach the detector, and many that do are still not registered. But since we not reach the detector, and many that do are still not registered. But since we
can expect to always lose track of a similar proportion of emitted light – perhaps can expect to always lose track of a similar proportion of emitted light – perhaps
90% – this does not matter much: we can expect all parts of the image to be 90% – this does not matter much: we can expect all parts of the image to be
similarly a↵ected, so relative di↵erences in brightness would still be reflected in
our pixel values. However, there are two more critical ways in which the images
Point Spread
Ruido
similarly a↵ected, so relative di↵erences in brightness would still be reflected in
our pixel values. However, there are two more critical ways in which the images
we can record are less good than the images we would like:
Function we can record are less good than the images we would like:

1. Uncertainty in space. Ideally, all light originating from a particular point 1. Uncertainty in space. Ideally, all light originating from a particular point
in the specimen would strike the detector in exactly the same place, and in the specimen would strike the detector in exactly the same place, and
 Formación de la imagen: PSF

• La intensidad detectada está afectada por la PSF.

• Se modela como la convolución de la image ideal con la PSF.

• Es una redistribución de la intensidad en la imagen (3D)

• La PSF depende del tipo de microscopio, longitud de onda detectada

y la apertura numérica (NA) del objetivo

• Tamaño de cientos de nm

• Es un fenómeno físico (difracción) de la luz, no (solamente) por

defectos de construcción de lentes.

• Determina la resolución de la imagen.

• Efectos en:

• Tamaño de estructuras

• Intensidades detectadas

• Número de estructuras
 Formación de la imagen: PSF

PSF adquirida con “perlas” PSF teórica para el

en microscopio widefield. mismo microscopio.
Un acercamiento simplificado

Fuera de
foco

En foco

Fuera de
foco
136
PSF
CHAPTER 15. BLUR & THE PSF

Figure 15.5: Ten slices from a z-stack acquired of a fluorescent bead, starting from
above and moving down to the focal plane. The same linear contrast settings have been
applied to each slice for easy comparison, although this causes the in-focus bead to appear
saturated since otherwise the rings would not be visible at all. Because the image is
(approximately) symmetrical along the z-axis, additional slices moving below the focal
plane would appear similar.

(a) George Biddell Airy (b) Airy pattern (c) Surface plot of Airy pattern
(1801–1892)

Figure 15.6: George Biddell Airy and the Airy pattern. (a) During his schooldays, Airy
had been renowned for being skilled ‘in the construction of peashooters and other such
devices’ (see http://www-history.mcs.st-and.ac.uk/Biographies/Airy.html). The
rings surrounding the Airy disk have been likened to the ripples on a pond. Although the
rings phenomenon was already known, Airy wrote the first theoretical treatment of it in
 Resolución

• Mínima distancia
140 a la cual dos puntos son discernibles.
CHAPTER 15. BLUR & THE PSF

(a) 2 disk radii separation (b) 1 disk radius separation (c) 0.8 disk radii separation
Rayleigh’s criterion

0.61Figure 15.9: Airy2 patterns

n separated by di↵erent distances, defined in terms of Airy
rx,y = disk radii.rzThe
= top row contains the patterns themselves, while the bottom row shows
2
NAfluorescence intensity
NA profiles computed across the centres of the patterns. Two distinct
spots are clearly visible whenever separated by at least one disk radius, and there is a dip
NA = n sin apparent
↵ in the profile. However, if the separation¿Qué factores
is less than afectan
one radius, la resolución?
the contrast
rapidly decreases until only one structure is apparent.
naire = 1, nagua = 1.34, naceite = 1.5
 pendix for sources) to secure needed excitation wavelengths and/or separation
of fluorescence emission wavelengths. Several of the commercial sources now

Fluorescence Microscope
also provide custom-made filters and a dichroic mirror, installed in a SINGLE
manufacturer-supplied cube, which are capable of handling double or triple fluo-
rochrome stained specimens without crossover problems (e.g. DAPI & FITC,
DAPI & FITC & TEXAS RED, etc.).

THE MICROSCOPE OBJECTIVE

In view of the low emitted light levels, the role of the objective in fluorescence
microscopy is crucial because it is the objective’s function to gather light from
the specimen. The angle of the cone of light accepted by the objective is deter-
mined by the numerical aperture (N.A.) of the objective. (Fig. W) In transmitted
light fluorescence microscopy, the intensity of the light reaching the eye or other
detector varies directly as the square of the numerical aperture of the objective
and the condenser and inversely as the square of the total magnification. On
the other hand, in reflected light fluorescence, the intensity of the image varies
directly as the fourth power of the numerical aperture of the objective in use, as
well as inversely as the square of the total magnification (I=N.A.4/Mag.²). This
difference comes as a result of the objective’s initially functioning as a condenser
for concentrating light on the specimen, and then as the usual light gatherer for
image formation. The implications are clear: high numerical aperture objectives
will yield images of higher intensity than will identical magnification objectives
of lower numerical aperture. For example, other things being equal, a 40X ob-
jective with a N.A. of 1.0 will yield images more than five times brighter than a
40X objective with a numerical aperture of 0.65. A further implication is that,
if possible, the employment of lower magnification visual eyepieces (e.g. 8X
magnification) will also increase the brightness of the image as compared to the
more commonly used 10X observation eyepieces. Additionally, in reflected light
fluorescence, the excitation light is concentrated by the objective (in its function
as condenser) only on the area being observed. As a result the intensity of the
excitation light is much higher than in transmitted light darkfield fluorescence
where the area being excited does not change as objectives are changed. Also
in darkfield, the numerical aperture of the objective must be reduced to below
that of the condenser to preserve the darkness of the field of view.

e and
ility

erture

x of
ront
n

Cone
by

PAGE 22 / THE MICROSCOPE OBJECTIVE

 PSF

Lateral
Axial

NA=0.8 NA=1.0 NA=1.2

Numerical Aperture and Image Resolution

http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
Ejercicio

500nm
 Ejercicio

500nm

200nm 20nm 2nm

132 CHAPTER 15. BLUR & THE PSF

(a) Sharp (b) Blurred

Figure 15.1: Schematic diagram showing the e↵ects of blur. Think of the sand as photons,
and the height of the sandcastle as the intensity values of pixels (a greater height indicates
more photons, and thus a brighter pixel). The ideal data would be sharp and could
contain fine details (a), but after blurring it is not only harder to discriminate details, but
intensities in the brighter regions have been reduced and sizes increased (b). If we then
 Ruido

• Fuentes de ruido

• Ruido de fotones: depende de la señal

• Ruido de adquisición: independiente de la señal, depende del detector

• En general
144 para reducir el ruido hay que aumentar la señal
CHAPTER (SNR).
16. NOISE

(a) Noisy image (b) Ideal image (c) Result of (a) - (b) (d) Histogram of (c)

Figure 16.1: Illustration of the di↵erence between a noisy image that we can record (a),
and the noise-free (but still blurred) image we would prefer (b). The ‘noise’ itself is what
would be left over if we subtracted one from the other (c). The histogram in (d) resembles
a normal (i.e. Gaussian) distribution and shows that the noise consists of positive and
negative values, with a mean of 0.

2. Noise is independent at each pixel – The value of the noise at any pixel does
 Características del ruido

• Aditivo

• Aleatorio

• Independiente en cada pixel

• Sigue una cierta distribución (variable aleatoria)

• Gaussiana (ruido de adquisición)

16.1. INTRODUCTION 145

• Poisson (ruido de fotones)

• Desviación estándar (potencia)

 Características del ruido

(a) Std. dev. 5 (b) Std. dev. 10 (c) Std. dev. 20

Figure 16.2: Gaussian noise with di↵erent standard deviations. (Top) Noise values only,
shown as images (with contrast adjusted) and histograms. (Bottom) Noise values added
to an otherwise noise-free image, along with the resulting histograms. Noise with a higher
 Fuentes de ruido

• Ruido de fotones

• Debido a la emisión y detección de la luz

• La potencia cambia con señal, cambia localmente en la imagen

• Sigue una distribución de Poisson

• Ruido de adquisición

• Debido a errores de cuantificación de fotones detectados

• La potencia de la señal es la misma en toda la imagen

• Sigue una distribución Gaussiana

Si!
• Mayor
156 exposición, mayor señal, ¿menos ruido?CHAPTER 16. p
NOISE
SNR = p =

(a) A noise-free signal (b) Photon noise (c) Read noise (d) Photon + Read
Señal y ruidos de
Señal sin ruido Señal y ruido fotones Señal y ruido adquisición
noise fotones adquisición

Figure 16.10: An illustration of how photon noise di↵ers from read noise. When both are
148 CHAPTER 16. NOISE

(a) Original image (b) Averaging 2 adjacent pixels (c) Averaging 9 adjacent pixels

Figure 16.3: Noise reduction by averaging adjacent pixels.

sian noise with standard deviations of 5. Estimate (and optionally test with
Image Calculator... and Measure), what the standard deviations will be:
 Fluoróforos

Violet laser Blue laser Yellow laser Red laser Far Red laser
(405 nm) (488 nm) (561 nm) (640 nm) (750 nm)

Stoke’s shift

http://docs.abcam.com/pdf/secondary-antibodies/abcam-fluorochrome-chart.pdf
 Tipos de microscopía de fluorescencia

• El tipo de microscopio define la PSF, ruido y resolución temporal.

• No
164 hay un tipo de microscopio
CHAPTERóptimo
17. en todos los aspectos.
MICROSCOPES & DETECTORS

Widefield LSCM SDCM 2 photons TIRF

(a) Widefield (b) LSCM (c) SDCM (d) 2-Photon (e) TIRF

Figure 17.1: Schematic diagrams to show the di↵erences in excitation patterns. Here, an
inverted microscope is assumed (the cell is resting on a coverslip, and illuminated from
below). During the recording of a pixel, light can potentially be detected if it arises from
any part of the green-illuminated region, although in the laser scanning and spinning disk
confocal cases the pinhole will only permit a fraction of this light to pass through. Note
that, for the 2-photon microscope, the excitation is confined to only the small, central
region, while the red light above and below is not capable of exciting the fluorophores
that have been used.

17.2 Types of microscope

 Widefield microscopy

• Toda la muestra es iluminada

simultáneamente.

• Todo los fluoróforos emiten juntos.

• Los fotones detectados provienen de

toda la muestra. (a) Widefield (b
• En una muestra gruesa hay mucho blur.

Figure
• Se captura la fluorescencia in-focus y out-of-focus
17.1: Schematic diagr
inverted microscope is assum
• Buena SNR (mucha señal).

below). During the recording

• Baja resolución espacial y detección de pequeñas
anyestructuras.

part of the green-illumin

• Relativamente rápida adquisición temporal. confocal cases the pinhole wi
that, for the 2-photon micro
region, while the red light a
that have been used.
 Laser Scanning Confocal Microscopy

• Optical sectioning.

• Se ilumina y detecta un pixel por vez.

• Laser en un volumen pequeño.

• Pinhole: poca luz out-of-focus.

• Mucha mejor resolución en z

Light source
• Resolución xy similar a (a)
widefield

Widefield (b) LSCM (

Aperture
• Adquisición lenta y ruidosa (en término de
fotones)
Figure 17.1: Schematic diagrams to show th
Light
inverted microscope is assumed (the cell is
Detector
Beam
splitter

below).
Mejor resolución During the recording of a pixel, ligh
espacial
Aperture

any
a cambio part of
de resolución
temporal y ruido.
the green-illuminated region, alth
confocal cases the pinhole will only permit a
that, for the 2-photon microscope, the exci
Focal plane

region, while the red light above and below

 Spinning Disk Confocal Microscopy

• Compromiso entre la velocidad de

widefield y resolución de LSCM

• Se adquieren varios pixeles no

adyacentes en simultáneo.

• Regiones lejanas para no mezclar la

intensidad out-of-focus.

(a) Widefield (b) LSCM (c) SDCM (d

• Logra optical sectioning mejorando la
resolución y SNR de widefield, e
gure 17.1: Schematic
incrementa la velocidaddiagrams to
show the di↵erences in e
de adquisición
frente a LSCM.
verted microscope is assumed (the cell is resting on a cov
elow). During the recording of a pixel, light can potentially
ny part of the green-illuminated region, although in the lase
nfocal cases the pinhole will only permit a fraction of this
hat, for the 2-photon microscope, the excitation is confine
gion, while the red light above and below is not capable
 Multiphoton Confocal Microscopy

• Para lograr la excitación del fluoróforo

es necesario la absorción simultánea
de múltiples (2) fotones.

• La región de excitación se reduce

considerablemente pues no logran
excitarse otras regiones.

•
(b) LSCM
Menor daño en la muestra.

(c) SDCM (d) 2-Photon (e

• Mayor penetración en la muestra
matic diagrams to
(cientos de um).
show the di↵erences
in excitation pattern
pe is assumed (the cell is resting on a coverslip, and illum
e recording of a pixel, light can potentially be detected if it
en-illuminated region, although in the laser scanning and s
pinhole will only permit a fraction of this light to pass thr
oton microscope, the excitation is confined to only the sm
red light above and below is not capable of exciting the fl
 Total Internal Reflection Fluorescence

• Se ilumina la muestra con un ángulo de

forma de excitar solamente la
superficie de la muestra.

• La adquisición es similar a widefield.

• Penetración menor a 100 nm.

CM (c) SDCM (d) 2-Photon (e) TIRF

to show the di↵erences in excitation patterns. Here, an

the cell is resting on a coverslip, and illuminated from
pixel, light can potentially be detected if it arises from
region, although in the laser scanning and spinning disk
ly permit a fraction of this light to pass through. Note
e, the excitation is confined to only the small, central
and below is not capable of exciting the fluorophores
 Super resolución

[Schermelleh, 2010]
Figure 2. Resolvable volumes obtained with current commercial super-resolution microscopes. A schematic 3D representation of focal volumes is shown
for the indicated emission maxima. The approximate lateral (x,y) and axial (z) resolution and resolvable volumes are listed. Note that STED/CW-STED and
3D-SIM can reach up to 20 µm into the sample, whereas PALM/STORM is usually confined to the evanescent wave field near the sample bottom. It should
be noted that deconvolution approaches can further improve STED resolution. For comparison the “focal volume” for PALM/STORM was estimated based
on the localization precision in combination with the z-range of TIRF. These indications do not necessarily constitute actual resolution as many other effects
(e.g., fluorophore orientation, local refractive index variations, flatfield quality of the camera, local aberrations, and statistical selection bias) influence
 Detectores

• Conversión fotones → electrones → voltaje → intensidad

• Dependen del tipo de microscopio

• Fotomultiplicadores (Photomultiplier Tube -PMT-)

• Sensores bidimensionales (CCD, CMOS)

 Fotomultiplicadores (PMT)
122 3 Fluorescence Microscopy

Photocathode Secondary
high voltage (−) electrons Anode
500–2000 V

Incident

To current-
to-voltage
photon

amplifier
Focusing Dynode
electrode

Figure 3.19 Schematics of a photomultiplier.

• Un “pixel” a la vez (LSCM)

tubes (PMTs) are not used in standard widefield fluorescence microscopes, but
• Conversión en el fotocátodo.

serve as light detectors in laser scanning confocal microscopes.

A PMT is a signal-amplifying device that exploits the photoelectric effect and a
secondary emission phenomenon, that is, the ability of electrons to cause emission
• Aceleración de electrones (emiten más electrones en varias etapas).

of other (secondary) electrons from an electrode in a vacuum tube. Light enters

a PMT through a quartz (or glass) window and strikes a photosensitive surface
• Baja Quantum Efficiency (conversión de fotones en electrones): ~15%

(a photocathode) made of alkali metals (Figure 3.19). The photocathode releases

electrons that subsequently strike the electrode (dynode), which releases a still
larger number of electrons. These electrons hit the next dynode. A high voltage
• Bajo ruido de adquisición, “alto” ruido de fotones (peor en baja
(1–2 kV) is applied between subsequent dynodes.
iluminación). The electrons are accelerated and the process is repeated leading to an ampli-
fication of the first electric current generated on the photocathode. The current
measured at the last dynode is proportional to the intensity of light that was
incident on the photocathode. The gain obtained by amplifying the electric current
through subsequent dynodes can be as high as 107 –108 . However, the voltage,
 Charged Coupled Device (CCD)

• Región dedicada a sensar fotones, con múltiples “bins”, en simultáneo.

Fotones son convertidos La carga es movida a un registro Se cuantifica y amplifica la

en electrones (carga). intermedio y libera la región de sensado. carga, fila por fila, bin a bin

• QE muy alta (~90%): más tiempo de exposición

• Ruido de adquisición es lo más problemático debido a la etapa de

amplificación.

• Pixel binning
 Electron Multiplying CCD (EMCCD)

Región de
sensado.

Multiplicación
de electrones Amplificador de salida

• “Un CCD con PMTs”: agrega una etapa de multiplicación de

electrones (PMT)

• Varias (>500) etapas de amplificación: aumenta el ruido de fotones a

costa de reducir ruido de adquisición.

• Alta velocidad.

• QE se reduce.
Complementary Metal Oxide Semiconductor (CMOS)

• Conversión fotón-electrón-voltaje en el “pixel”

• Acceso a cada “pixel” independientemente (window of interest).

• Más rápido que CCD

• Regiones de “pixel” más pequeños (menor luz): más ruido.

• QE menor que CCD.

 Scientific CMOS (sCMOS)

• Similar a CMOS pero con

• mayor tamaño de sensor

• menor ruido adquisición (no hay multiplicación)

• alta velocidad

• Precio
 ANDOR iXon Ultra 888 ANDOR Zyla 4.2 PLUS

Key Specifications
Active pixels (HxV) 1024 x 1024 2560 x 2160 2048 x 2048
Pixel size (WxH; um) 13 x 13 6.5 x 6.5 6.5 x 6.5
Image area (mm) 13.3 x 13.3 13.3 x 13.3 16.6 x 14.0
Pixel Well Depth (e-) 80,000 30,000 30,000
Frame rates (fps) 26* - 96901 40* - 100*2 53* - 101*2
Quantum Efficiency (max) 90% 60% 82%
Price ??? ? ?

* Full frame
1 93 fps (512 x 512), 697 fps (128 x 128)
2 USB 3.0 o Camera Link 10-tap
 490 fotones 68 fotones 8 fotones
por pixel por pixel por pixel
sCMOS (Zyla 5.5)
EMCCD (iXon Ultra 888)
Microscopía electrónica
 Microscopio electrónico

h h 1
= =p q
p 2m0 eU 1 + eU 2
2m0 c

Longitud de onda y resolución dado el voltaje

U(kV) 80 100 200 300

λ (Å) 0.0418 0.0370 0.0251 0.0197

r (Å) 2.5555 2.2652 1.5348 1.2048

Mass of an electron = 9.1091x10-31 kg

Speed of light = 299,790,000 meters/second
Charge of an electron = 1.602x10-19 coulombs
Planck's Constant = 6.6256x10-34 m2kg/sec
 http://www.microbiologyinfo.com/wp-content/uploads/2015/04/Light-Microscope-and-Electron-Microscope.png
Óptico vs. Electrónico
 Óptico vs. Electrónico

• Partículas: fotones / electrones.

• Preparación es más exigente y larga en ME.

• No se pueden analizar muestras vivas en ME por el vacío dentro del

microscopio.

• Lentes y condensadores son electromagnéticos en ME.

• Mucha mayor resolución (200nm y 0.1nm) y magnificación (varios cientos

40 de THREE-DIMENSIONAL
veces mayor) en ME.

ELECTRON MICROSCOPY

• Mayor exigencia en condiciones de uso (voltaje, enfriamiento, …),

mantenimiento y entrenamiento en el ME.

• Una sola longitud de onda (no hay colores) en ME.

• PSF y CTF (Contrast Transfer Function)

[Frank, 2006]
 Microscopio electrónico

Getting Started in Cryo-EM with Professor Grant Jensen

[Frank, 2006]

re 2.1 Schematic diagram of a transmission electron microscope. Adapted from a

wing provided by Roger C. Wagner, University of Delaware (Web site: www.udel.edu/
ogy/Wags/b617/b617.htm).

1. Principle of the Transmission Electron Microscope

transmission electron microscope uses high-energy (100 keV or higher)

trons to form an image of very thin objects.1 Electrons are strongly scattered

s section is not meant as a substitute for a systematic introduction, but serves mainly to provide a
ral orientation, and to introduce basic terms that will be used in the text.

15
 Referencias

• Burger, W., & Burge, M. J. (2009). Principles of Digital Image Processing. London:
Springer London. doi:10.1007/978-1-84800-191-6

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