Teórico Nº15

2023
Microscopía II
Biologia Celular y Molecular
Facultad de Farmacia y Bioquímica
Universidad de Buenos Aires

Prof. Dr. Nicolás O. Favale

Tipos de Microscopía Óptica

Tipos de Microscopia
Tejidos
Microscopia Óptica [
Células

Sin marcación Con marcación

Observación directa Colorantes

Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Proteínas Celulas vivas
Contraste de Fases
interferencial
(DIC - Nomarski)
terFluorescencia
a given time (Figure 1). Photoluminescent processes that are generated through excitation,
hether this is via physical, mechanical, or chemical mechanisms, can generally be subdivided into
uorescence and phosphorescence. Fluorescencia
Figure 1. Fluorescence
Fotoluminiscencia principle.
que se extingue Schematic
al cesar la radiación que lorepresentation of científico
provoca (Vocabulario the fluorescence
y técnico RACEFN)

phenomenon in the classical Bohr model. Absorption of a light quantum (blue) causes an
Fotoluminiscencia que caracteriza a las sustancias que son capaces de absorber energía en forma de
electron to
radiaciones move to a higher
electromagnéticas y luegoenergy orbit. de
emitir parte After
esa residing
energía eninforma
this de
“excited
radiaciónstate” for a
electromagnética
particular
de longitudtime, the diferente
de onda fluorescence
. lifetime, the electron falls back to its original orbit and the
fluorochrome dissipates the excess energy by emitting a photon (green).

Hellen C. Ishikawa-Ankerhold et al Molecules 2012, 17, 4047-4132

Fluorescencia

Fluorescencia
Absorción - Desexitación no radiativa - Emisión

Tiempo- nanosegundos por los que puede considerarse instantáneo

Fluorescencia
Fosforescencia
Fluorescencia

Fluorescencia

Absorción - Desexitación no radiativa - Emisión

Fluorescencia

Fluoróforos
Varios marcadores en forma simultánea, y puede observarlos por separado

Exitación Emisión
Fluorescencia

Moléculas Orgánicas
Generalmente son moléculas orgánicas

Rodamina
DAPI

FITC
Alexa 488

Cy5
Quantum dot

Puntos Cuánticos
Mayor vida media que las moléculas orgánicas

Molecules 2012, 17, 4047-4132

http://www.plasmachem.com
Fluoroforos

¿Cómo darle especificidad / selectividad?

F a) Fluorocromo con selectividad

F RNA/DNA b) Fluorocromo unido sonda hibridación

F Anticuerpo c) Fluorocromo unido anticuerpo

F Afinidad d) Fluorocromo unido molécula que le da

especificidad/selectividad
Fluoroforos - especificidad/selectividad

Fluorocromo con selectividad

Hoechst

Eur. J. Biochem. 222, 721 -726 (1994)

0 FEBS 1994

Three-dimensional crystal structure

of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed
with the minor-groove-binding drug Hoechst 33258
M. Cristina VEGA’ *, Isabel GARCiA SAEZ’ ’, Joan AYMAMi*, Ramon ERITJA’, Gijs A. VAN DER MAREL3,
Jaques H. VAN BOOM3, Alexander RICH4 and Miquel COLL’
Departament de Biologia Molecular i Cel.lular, Centre d’Investigaci6 i Desenvolupament-C. S. I. C., Barcelona, Spain
* Departament d’Enginyeria Quimica, Universitat Politttcnica de Catalunya, Barcelona, Spain
Fluoroforos - especificidad/selectividad

Fluorocromo unido a moléculas

Faloidina-(FITC) F Afinidad

Toxina Fluorescente
F-actina verde

Favale et al JLR (2015)

Fluoroforos 2002). In the following
paragraphs the available mFISH
- especificidad/selectividad of ap
banding probe sets are listed according to their quality of 4
resolution. Fluorocromo unido sonda Cost
1. The cross-speciesFISH color banding (Rx-FISH) or band
Harlequin-FISH probe set
(Fluorescence (Fig.Hybridization)
In-Situ 2) provides the lowest band
resolution of 80-90 bands per haploid human karyotype on th
F RNA/DNA 5
tech
libra
hum

T. Liehr, H. et al Multicolor FISH probe sets and their applications

Fig. 1. 25-color FISH karyogram of a normal female metaphase (Mrasek
Histol Histopathol (2004) 19: 229-237
Fluoroforos - especificidad/selectividad

Fluorocromo unido anticuerpo

Inmunofluorescencia

F Anticuerpo
Fluoroforos - especificidad/selectividad

Fluorocromo unido anticuerpo

Inmunofluorescencia

F Anticuerpo

NO Favale, MC Fernandez-Tome, LG Pescio, and NB Sterin-Speziale

Biochim Biophys Acta, Nov 2010; 1801(11): 1184-94
Ejemplo de uso de M. Fluorescencia

Localización intracelular
control Tr1-34 Tfx tratadas

marcador de nucleo
anticuerpo anti-Tfx
anticuerpo anti-R

control
2rio
Rojo FITC
1rio

Verde Tfx
Ejemplo de uso de M. Fluorescencia

Localización intracelular
control Tr1-34 Tfx tratadas

marcador de nucleo
anticuerpo anti-Tfx
anticuerpo anti-R

control tratadas Tr1-34

Rojo

?
Verde

superposición superposición
Ejemplo de uso de M. Fluorescencia

Localización intracelular
Sphingosine kinase
Sphingosine Permeabilización
and epithelial
kinasecelland
homeostasis
epithelial cell hom

Fluorocromo unido Fluorocromo unido

Fluorocromo con Proteína Fluorescente /
selectividad
molécula que le da anticuerpo
Inmunofluorescencia Fijación
especificidad/selectividad Inmunofluorescencia

Santacreu BJ et al (2019) PLoS ONE 14(3): e0213917.

Tipos de Microscopía Óptica

Tipos de Microscopia
Tejidos
Microscopia Óptica [
Células

Sin marcación Con marcación

Observación directa Colorantes

Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Proteínas Celulas vivas
Contraste de Fases
interferencial
(DIC - Nomarski)
Proteínas Bio-luminicente
Aequorea victoria
GFP
(green fluorescent protein)
The Nobel Prize in Chemistry 2008
Osamu Shimomura, Martin Chalfie, Roger Y. Tsien

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The Nobel Prize in Chemistry 2008 11 laminas beta

centro crómoforo

The Nobel Prize in Chemistry 2008

Osamu Shimomura, Martin Chalfie, Roger Y. Tsien

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Share MorePhoto:
Share U. 18
18 Share Montan
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Osamu Shimomura
The NobelPrizePrize
share: 1/3 in Chemistry 2008

Photo: U. Montan Photo: U. Montan Photo: U. Montan

Osamu Shimomura Martin Chalfie Roger Y. Tsien
Prize share: 1/3 Prize share: 1/3 Prize share: 1/3
"The Nobel Prize in Chemistry 2008". Nobelprize.org. Nobel Media AB 2014. Web. 7 Sep 2014.
The Nobel Prize in Chemistry 2008 was awarded jointly Friday
to Osamu Shimomura,
Harbor en la IslaMartin Chalfie
San Juan and
(Washington, Estados Unidos),
Roger Y. Tsien "for the discovery and development of the green fluorescent protein, GFP".
Marcadores de Organelas

Diferentes Proteínas Fluorescentes

Hellen C. Ishikawa-Ankerhold et al Molecules 2012, 17, 4047-4132

http://www.olympusmicro.com
Experiencia
GFP
Proteínas Quiméricas

Transfección

Producir una proteína quimérica

y realizar fluorescencias Secuencia GFP poliA ProteínaX GFP
en células vivas

Selección Promotor
ORI
Procarionte Eucarionte

ORI
Poliligador

G418 Selección
Eucarionte ORI
 Experiencia

Hind III
Kpn I
BamH I
BstX I
EcoR I
EcoR V
BstX I

7034 pb
Not I
Xho I
Xba I
Apa I
Actina-Cherry

Hind III
Kpn I
BamH I
BstX I
EcoR I
EcoR V
Actin

BstX I
7034 pb

Not I
Xho I
Xba I
Apa I
Experiencia
Actina-Cherry

Competentes sin transformar Competentes transformadas Competentes transformadas

LB + Ampicilina LB sin Ampicilina LB + Ampicilina
Experiencia

GFP
Receptor-GFP DIC

¿Cual es su localización?
Proteínas Quiméricas
Aplicar
GFP

Transitoria
DIC GFP Superposición

Estables G418
DIC GFP Superposición
Seguimiento en células vivas
GFP
(green fluorescent protein)

Molecular Biology of the Cell

Vol. 11, 1213–1224, April 2000

Promotor-GFP
Axonal Membrane Proteins Are Transported in
Neuronas Distinct Carriers: A Two-Color Video Microscopy
Study in Cultured Hippocampal Neurons□ V

Christoph Kaether,* Paul Skehel,† and Carlos G. Dotti*‡

*European Molecular Biology Laboratory, Cell Biology Program, 69012 Heidelberg, Germany; and
†
Organismos transgénicos
GFP
(green fluorescent protein)
Organismos transgénicos

Starfire Red®
Cosmic Blue®
Electric Green®
Galactic Purple®
Sunburst Orange®
Moonrise Pink™

We invite you to snorkel around the site and get to

Tipos de Microscopía Óptica

Tipos de Microscopia
Tejidos
Microscopia Óptica [
Células

Sin marcación Con marcación

Observación directa Colorantes

Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Proteínas Celulas vivas
Contraste de Fases
interferencial
(DIC - Nomarski) Microscopios

Convencional (widefield)
FRET
Confocal [
FRAP
Multifotónico
Epifluorescencia

¿Que es la Epiiluminación?

Entonces
¿Cómo se soluciona?
Epifluorescencia

¿Que es la Epiiluminación?
Epifluorescencia

Microscopio de Epifluorescencia
detector

ocular
Filtro excitación
Filtro emisión
Hg

Espejo dicroico
Cubo

objetivo
Epifluorescencia

Microscopio de Epifluorescencia
detector

ocular
Filtro excitación
Filtro emisión
Hg

Espejo dicroico
Cubo

objetivo
Epifluorescencia

Microscopio de Epifluorescencia

Hg

CUBO
Tipos de Microscopía Óptica

Tipos de Microscopia
Tejidos
Microscopia Óptica [
Células

Sin marcación Con marcación

Observación directa Colorantes

Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Proteínas Celulas vivas
Contraste de Fases
interferencial
(DIC - Nomarski) Microscopios Deconvolución

Convencional (widefield)
FRET
Confocal [
FRAP
Multifotónico
Microscopia
Microscopia

Anológico ✔
Mejorar por dos métodos
Digital
Microscopía Confocal
Confocal
Foco en la luz incidente (Laser)
Barrido Secuencial
Foco de la luz emitida
Microscopía Confocal
Confocal
Elimina la luz Fuera de Foco

Convencional Confocal
Microscopía Confocal
Confocal

Seccionamiento Óptico

Análisis de diferentes
zonas de la célula

Reconstrucción de los
Eje XZ - YZ

Reconstrucción de célula
en tres dimensiones
Microscopia
Microscopia
Confocal
Seccionamiento Óptico- Eje Z (perfil)

NO Favale, MC Fernandez-Tome, LG Pescio, and NB Sterin-Speziale

Biochim Biophys Acta, Nov 2010; 1801(11): 1184-94
Microscopia
Confocal
Seccionamiento Óptico - 3D
Multifotón vs Confocal
Un paso mas allá del Confocal
Confocal Multifotónico

Luz incidente tiene profundidad limitada Fotones de menor energia (IR)

Sigue existiendo luz fuera de foco Fotones coinciden en un punto (femtosegundos)

488nm

960nm 960nm
Multifotón vs Confocal
Un paso mas allá del Confocal
(a) Point-scanning confocal Two-photon excitation (c)
microscopy microscopy Objective lens of m

Confocal
Electron excited state
Multifotónico
Immobilize
Excitation
mouse
photon 2
Excitation Emission (960 nm) Emission
photon photon photon
(488 nm) (507 nm) (507 nm)
Excitation
photon 1
(960 nm)

Electron ground state

(d)

(b) Excitation Excitation

photon photons
(488 nm) (960 nm)

Focal
plane Emission
photons
at focal
plane
(507 nm)
Multifotón vs Confocal
Un paso mas allá del Confocal

Confocal Multifotónico

http://www.olympusmicro.com
Tipos de Microscopía Óptica (960 nm) (b) and a new discussio
Objective lens of microscope
focuses on the cell-
Tipos
Electron groundde Microscopia
state leads to an entirely
Immobilized

Intra vital which explores th

mouse

underlying synaptic

(b)
Objective lens of microscope
(c) Increased Clarity
As experienced teac
ate students, we are
derstanding. Being
Immobilized can have a profoun
mouse lecular processes wi
updated many of th
and added models
standing. From the
FIGURE 4-21 Two-photo excitation microscopy allows
to distancia
theabsorption
conservation
deep penetration for intravital imaging. (a) In conventional
point-scanning confocal microscopy, of a single

In two-photon excitation, strength

two lower-energyof tropomy
photon results in an electron jumping to the excited state.
de trabajo photons arrive
almost instantaneously and induce the electron to jump to
the excited state. (b) Two-photonthese figures
microscopy can be usedcomm
to observe cells up to 1 mm deep within a living animal
(c) immobilized on the microscope lar stage.
structure
(c) Neurons inthata lobsterca
were imaged using two-photon excitation microscopy.
alone.
[Part (c) unpublished data from Peter KloppenburgIn conjunctio
and Warren R. Zipfel.]

icons have been rev

viii PREFACE
allowing students a
Microscopia

Anológico
Mejorar por dos métodos
Digital ✔
Análisis digital
Convolución
es un operador matemático que transforma dos funciones f y g en una tercera función que en cierto
sentido representa la magnitud en la que se superponen f y una versión trasladada e invertida de g

3ra función

función f
función g

La imagen obtenida es un producto

de la imagen original mas la convolución
Análisis digital
Deconvolución

Convolución

? Conocer

Deconvolución

PSF
(Point spread function)
Función de dispersión de un punto
Análisis digital
Disco de Airy
PSF
(Point spread function)
Función de dispersión de un punto

Analizar lo que le ocurre a una Unidad de Imagen Establecer función (Algoritmo matematico)
“Conocida” Para una condición determinada
Análisis digital
Deconvolución

http://www.olympusmicro.com
Tipos de Microscopía Óptica

Tipos de Microscopia
Tejidos
Microscopia Óptica [
Células

Sin marcación Con marcación

Observación directa Colorantes

Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Proteínas Celulas vivas
Contraste de Fases
interferencial
(DIC - Nomarski) Microscopios Deconvolución

Convencional (widefield)
FRET
Confocal [
FRAP
Multifotónico
FRAP-FRET
Técnicas en células vivas
FRET
Fluorescence resonance energy transfer

(a) (a)
433 nm 475 nm 433 nm FRET 530 nm 433 nm

CFP YFP CFP YFP C

X Y X Y
L
(b) d

5 nm phosphor
sub

(a) (b)
530 nm 433 nm 475 nm 433 nm 530 nm 0.0
FRET

Protein
CFP YFP CFP YFP
kinase Interacción Entre
Protein
phosphatase P
e Inter proteinas
Ligand Sensor
domain domain 10 µm EXPERIM
(binds (substrate local bioc
phosphorylated for kinase) protein con
substrate) FIGURE 424 Protein-protein interactions can be visualized to the envi
by FRET. The idea behind FRET is to use two different fluorescent consists of
(b) Time (minutes)
proteins so that when one is excited, energy will be transferred to the sequence t
second one by FRET, provided that they are sufficiently close. (a) In this
FRAP-FRET
CFP YFP CFP YFP
Técnicas en células vivas
X Y FRET X Y
Fluorescence resonance energy transfer
(b)

ph

(b)

10 µm EX
loc
pro
FRAP-FRET
Técnicas en células vivas
FRAP
Fluorescence recovery after photobleaching

Velocidad de difusión

Velocidad de transporte

Compartamentalización
Indicadores de iones
Técnicas en células vivas
Indicadores
Tipos de Microscopía Óptica

Tipos de Microscopia

Microscopia Óptica Microscopia Electrónica

Sin marcación Con marcación

Colorantes
Observación directa Fluorescentes
Enzimas
Contraste de Fases Moléculas Orgánicas

Campo Oscuro Semiconductores

Contraste de Fases
interferencial
(DIC - Nomarski)
Microscopia

Microscopía Óptica

Tipos de Microscopía

Microscopía Electrónica ✔
Aumentando la resolución Microscopio electrónico

Microscopia Electrónica
Aumentando la resolución Microscopio electrónico

d0 = 0,61 . 𝜆 / AN

aplica para cualquier

onda electromagnética

Microscopia Óptica 380-750 nm do= 200 nm

Microscopia Electrónica 0,002 nm doteorico= 0,001 nm doreal= 0,1 nm

Microscopia

Microscopía Óptica

Tipos de Microscopía

Microscopía Electrónica ✔

doreal= 0,1 nm
Aumentando la resolución Microscopio electrónico

Microscopio Electrónico de Transmisión

(MET o TEM )

Microscopia Óptica Microscopia Electrónica

VACIO

200 nm 0,1 nm
 grid, but is excluded from the regions where the sample
has adhered. When we view the sample in the TEM, we see over
Muestras where the stain has been excluded, so the sample is said to and
be negatively stained. Because the stain can precisely reveal were
beau
Preparación de las muestras
the topology of the sample, a high-resolution image can be
obtained (Figure 4-29c). led t
Samples can also be prepared by metal shadowing orga
T
Fijar (GA y TO4Os) (Figure Crioelectrónico
4-30). In this technique, (cryo-EM)
the sample is absorbed to
the s
a small piece of mica, then coated with a thin film of plati-
num by(Congelado
evaporation of alta presión-etano)
the metal, then dissolved with acid that
Deshidratar or bleach, leaving the platinum coating (known as a replica). abou
The platinum coating can be generated from a fixed angle samp
Secciones 50-100 nm Nobel 2017
or at a low angle as the sample is rotated, in which case it is and
Dubochet,
called low-angle Frank, Henderson
rotary shadowing. When the replica is trans- in th
ferred to a grid and examined in the TEM, it provides infor- tissu
Contrastar (Os, Pb y U) - electrodenso here
mation about the three-dimensional topology of the sample.
Réplica (sombreado)
(a) (b)
Sample Mica surface

e- e- 1

Evaporated platinum

Metal replica

Evaporated carbon

Carbon film

Metal replica ready

for visualization
4
 take place in the cytosol, where 2 ATP molecules per glucose branes t
Microscopia electrónica
molecule are generated. The terminal stages of oxidation and an exte
Preparación de las muestras limited
Outer form di
Inner membrane Cristae membrane are emb
thylako
and oth
that gen
is used t
diates b
are then
The
mitocho
in Chap
chlorop
mon: bo
and bot
3 m the key
Intermembrane encoded
space Matrix Matrix sized on
granules
the prot
FIGURE 120 Electron micrograph of a mitochondrion in a pan- and are
creas cell. The smooth outer membrane forms the outside boundary incorpo
 biological material by electron microscopy. The most widely
Microscopia electrónicaused instrument is the transmission electron microscope,
but also in common use is the scanning electron microscope
Tipos de Microscopia eletrónica
(SEM), which provides complementary information, as we
(a)

discuss at the end of this section (Figure

Microscopio Electrónico de Transmisión
4-28, right).
Microscopio Electrónico de Barrido
(MET o TEM) (MEB o SEM )
TEM SEM
Tungsten filament
Filamento Tungsteno
(cathode)
Anode
Anodo 3 mm

Condensador
Condenser lens
(c)

Beam
Haz deofelectrones
electrons

Scanning
Deflector
coils
Specimen
espécimen
Lente
Electromagnetic
objetivo
objective lens
electromagnéticas

Lente
proyección
Projector lens
electromagnéticas

0,1 nm 10 nm

Detector
Detector
Specimen
espécimen
 than that of transmission instruments.
Aumentando la resolución Microscopio electrónico Figure 1-12a)
LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE
menting their
clearly reveal
KEY CONCEPTS OF SECTION 4.3
CYTOSOL Figurethis,
9–52 it is nuclea
The neces
frozen and identify
nuclear envelopean
Electron Microscopy: High-Resolution a high-resolution
this reason,SEM,me
Imaging field emission gun as the
These were develop
views of each sid
Electron microscopy provides very high-resolution imagesrepresentLysosomes,
the limit of resf
logical
because of the short wavelength of the high-energy electrons(compare molecu
with Figure 12
Martin Goldberg and Ter
used to image the sample.
nuclear Lysosomes ha
pore
was discovered
NUCLEUS

50 nm
Some of this non-protein-coding DNA probably regu-
being plunged into a coolant. A special sample holder keeps this hydrated speci-
lates expression of genes that make us uniquely human.
men at –160°C in the vacuum of the microscope, where it can be viewed directly
Connective Indeed, fish and humans have about the same number of
without fixation, staining,
tissue or drying. Unlike negative staining, in which what we
protein-coding
see is the envelope of stain exclusion around the particle, genes—about 20,000—yet as noted above,
hydrated cryoelectron
Cilia
microscopy produces an image from the the human genome isstructure
macromolecular over twice theHow-
itself. size of that in fish (see
ever, the contrast in this image is veryTable low, and1-2).
to The human
extract brain can amount
the maximum perform complex mental
MBoC6processes
of structural information, special image-processing m9.51/9.52suchtechniques
as reading must
and writing
be used, a as
textbook. Somehow
we describe next. these 20,000 human genes are exquisitely regulated such
that humans produce a brain with about 100,000,000,000
Multiple Images Can Be Combined neurons, which communicate
to Increase Resolution with one another at about E
100,000,000,000,000 interaction sites termed synapses.
LumenAs we saw earlier (p. 532),Goblet noise is important in light microscopy
Genomics—the study at
oflow
thelight
entirelevels, WHAT WE
DNA sequences of or-DON
but it is a particularly severe cell problem for electron microscopy of unstained mac-
ganisms—has shown us how close humans really are to our
romolecules. A protein molecule can tolerate a dose of only a few tens of electrons
nearest relatives, the great apes (Figure 1-26). •Human We know in detail a
DNA
per square nanometer without damage, and this dose is orders of magnitude
is 99 percent identical in sequence to that ofprocesses, chimpanzees such as D
below what is needed to define an image at atomic resolution. and transcription and
and bonobos; the 1
The solution is to obtain images of many identical molecules—perhaps tens
percent difference is about 3,000,000
but will we ever be ab
of thousands of individual images—and basecombine
pairs, but
themit somehow
to produce explains
an the obvious
averaged suchdifferences
rapid molecular
Smooth between our species,
Endothelium
image, revealing muscle
structuralBasaldetails that are hidden by the such
noiseasinthe
theevolution
original of action
human in brains
cells?
images. This procedure lamina during the past
is called single-particle 5,000,000 years
reconstruction. sincecom-
Before we last shared a com-
bining all the individual images, however, mon they
ancestor.
must be aligned with each other. • Will we ever be able
10 !m
Sometimes
FIGURE 125 All organs are organized it is possible
arrangements Genomics coupled with
to induce proteins and complexes to form crystalline
of vari- paleontological intracellular
findings structures
indi-
of the electron micros
arrays,
ous tissues, as illustrated in this in which
cross section of aeach artery is heldcates
smallmolecule in thethat
samehumans
orientationandinmice descended
a regular lattice. from a common
mammalian cells?
(arteriole). Blood flows through In
thethis case,
vessel the which
lumen, alignment
is linedproblem
by is easily solved, ancestor
and several that probably
protein lived about
structures 75 million
Tipos de Microscopía Óptica

Tipos de Microscopia
• Magnificación
Microscopia Óptica • Resolución Microscopia Electrónica
• Contraste

Sin marcación Con marcación

Colorantes Microscopio Electrónico de Barrido

Observación directa Fluorescentes (MEB o SEM )
Enzimas
Contraste de Fases Moléculas Orgánicas Microscopio Electrónico de Transmisión
(MET o TEM)
Campo Oscuro Semiconductores

Contraste de Fases Proteínas Celulas vivas

interferencial
(DIC - Nomarski) Microscopios

Convencional (widefield)
FRET
Confocal [
FRAP
Multifotónico
Muchas Gracias