1- Microscopia

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Biologia Celular y Molecular
Facultad de Farmacia y Bioquímica 
Universidad de Buenos Aires
Teórico	Nº1	
2022		
Microscopía	
Prof.	Dr.	Nicolás	O.	Favale
Un poco de historia…
- Robert Hooke (1635-1703)
“Célula”
Micrographia 
?
compuesto
- Antonie van Leeuwenhoek (1632-1723) 
espermatozoide
“corpusculos rojo”
Bacterias 
simple
Pasteur (1822-1895) 
“no contesto pero lo 
hizo contestable”
x200
 Microscopia
Tipos de Microscopía
Microscopía Óptica
Microscopía Electrónica
✔
0,2 µm
Límite de 
difracción 
 de Abbe
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Básicamente la función del microscopio ópticos es obtener 
una imagen magnificada, para así facilitar la observación de 
los detalles que escapan a la simple observación. 
Lente convergente o biconvexa
Avances en Microscopia
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How the optical microscope became a nanoscope
Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having bypassed a 
presumed scientific limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres. 
Using the fluorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can 
observe disease-related proteins aggregate and they can track cell division at the nanolevel.
Single-molecule microscopyStimulated Emission Depletion (STED) microscopy 
 Avances
 Microscopia Óptica
Microscopía Óptica 
Microscopio Microscopio Invertido
Partes del microscopico
Partes de un microscopio
Objetivo
Condensador
Fuente luz
Iris 
Retina 
Ocular
Detector
Espécimen
Ocular
Revolver 
Estativo 
Ocular
Objetivo
Condensador
Diafragma 
Apertura 
Condensador 
Tornillo
condensador
Tornillo
macrométrico
Tornillo
micrométrico
Fuente luz
Diafragma 
Campo 
Iluminación 
Platina 
Tornillo
X-Y
Conceptualmente un microscopio
Procesamiento de la información
Interacción 
• Luz-Luz 
• Luz-Materia
Fundamento de la microscopia
Parametros Básicos
• Magnificación 
• Resolución 
• Contraste
Fuente de iluminación 
Espécimen 
Camino óptico
Receptor
Iris 
Retina 
Luz - radiaciones electromagnéticas
Dos modelos se usan para describir a las radiaciones electromagnéticas: Modelos 
de Onda y Modelo Partícula
Propagación en el espacio de la Onda Electromagnética (vibran perpendicular). 
modelo de partícula modelo de onda
fotones
Parametros de la onda
Longitud de onda, de una onda sinusoidal, es el período espacial de la onda. La distancia 
sobre la cual se repite la forma de la onda, o la distancia entre dos puntos de una misma 
fase (ej: dos máximos)
𝜆= distancia = metro (µm o nm)
Frecuencia es una magnitud que mide el número de repeticiones por unidad de tiempo de 
cualquier fenómeno o suceso periódico. Es es la inversa del período temporal de la onda.
1/T = ν = evento. tiempo-1 = 1/s = Hertz (Hz)
Período espacial/temporal
modelo de onda
espacio
longitud de onda (𝜆) periodo (T)
E = h.v
E = h.c/𝜆
c = 𝜆 . v
Parametros de la onda Período espacial/temporal
https://iie.fing.edu.uy/proyectos/esopo/eem/
 1 
Digitalización I 
De la señal a la imagen 
El advenimiento de nuevas tecnologías en lo que respecta al procesamiento y análisis de imágenes 
ha abierto un nuevo campo en la microscopia. En los últimos años, el estudio de las imágenes 
digitales de microscopia no solo ha permitido un análisis más acertado y objetivo, sino que también 
ha posibilitado análisis estadísticos y cuantitativos a fenómenos que sólo eran reportados en forma 
cualitativa, es decir, sujeta a la interpretación del observador. Estas nuevas tecnologías parten de 
la base de la digitalización de las imágenes. Sin embargo, para comprender la digitalización y su 
funcionamiento, resulta importante comprender el más antiguo y eficiente de los dispositivos, la 
visión humana. 
 
El ojo humano como dispositivo de detección a emular…o no. 
Si bien este capítulo no pretende realizar una descripción anatómica/fisiológica exhaustiva del ojo 
humano, es importante comprender mínimamente su funcionamiento. Este conocimiento es 
necesario para conocer las ventajas y limitaciones que presenta este magnífico órgano humano. 
Siempre debemos recordar que la visión humana posee un fuerte sesgo dado por la propia 
Figura D1 Ondas electromagnéticas. Representación esquemática que compara el espectro visible detectado por la 
visión humana en el contexto de los diferentes tipos de ondas electromagnéticas. 
Interacción Luz-Luz y Luz-Materia
reflectada
Transmitida 
absorción 
Emisión Fluorescencia
refracción 
(medio)
dispersión 
(𝜆)
difracción interferencia
dispersión 
esparcimiento 
(scattering)
Luz-Luz
Parametros Básicos
• Magnificación
• Resolución 
• Contraste
8
CFI Plan Apochromat 
10XC Glyc
Compatible with a wide range of 
immersion media and clearing agents
In addition to water and immersion oil, this objective lens 
is compatible with a variety of tissue clearing agents. The 
lens also features chromatic aberration correction over a 
broad spectral range and is compatible with the Ti2 inverted 
microscope.
• NA: 0.50, WD: 5.50 mm (upright) / 2.00 mm (inverted)
• Chromatic aberration correction: from visible to IR
• High-transmittance Nano Crystal Coat
• Correction collar for spherical aberration correction
Cleared Tissue Imaging
Zebrafish larva
Plan Apo
60x/1.40 Oil
DIC
∞/ 0.17 WD 0.15
medio de inmersión
Código de color
de magnificación
Código de color
Correcciones óptica
Medio de inmersión
Magnificación
Diseño especializado
Distancia de Trabajo (WD)
Apertura numérica (NA)
Rosca
Lente
Espesor de cubreobjetos
Longitud del tubo
Interacción Luz-Materia /Luz-Luz
 Microscopia Óptica
La parte fundamental del microscopio 
Objetivo
Condensador
Fuente luz
Iris 
Retina 
Ocular
Detector
Espécimen
 Microscopia Óptica
La parte fundamental del microscopio 
8
CFI Plan Apochromat 
10XC Glyc
Compatible with a wide range of 
immersion media and clearing agents
In addition to water and immersion oil, this objective lens 
is compatible with a variety of tissue clearing agents. The 
lens also features chromatic aberration correction over a 
broad spectral range and is compatible with the Ti2 inverted 
microscope.
• NA: 0.50, WD: 5.50 mm (upright) / 2.00 mm (inverted)
• Chromatic aberration correction: from visible to IR
• High-transmittance Nano Crystal Coat
• Correction collar for spherical aberration correction
Cleared Tissue Imaging
Zebrafish larva
Plan Apo
60x/1.40 Oil
DIC
∞/ 0.17 WD 0.15
medio de inmersión
Código de color
de magnificación
Código de color
Correcciones óptica
Medio de inmersión
Magnificación
Diseño especializado
Distancia de Trabajo (WD)
Apertura numérica (NA)
Rosca
Lente
Espesor de cubreobjetos
Longitud del tubo
• Magnificación
• Resolución 
• Contraste
 Magnificación
Magnificación
Cuantas veces más grande es la imagen generada en 
comparación con el objeto 
 256 
 
 
ésta (2f), podemos ver que la marcha de rayos dari 
como resultado una imagen real (ya que podrta se 
proyectada sobre una superficie), invertida (vemos que 
esti apuntado hacia la direcciyn contraria del objeto 
original) y magnificada (es más grande que el objeto 
original) (Fig. 24-9 Imagen A). Esto es lo que ocurre en 
el microscopio por acciyndel objetivo. Ahora si a este 
sistema yptico le agregamos oculares, la resultante seri 
una imagen invertida-virtual, y con una mayor 
magnificaciyn (Fig. 24-9 Imagen B). Vemos que la 
imagen es virtual ya que espacialmente se observari 
pryxima a la base del microscopio (aproximadamente a 
25 cm). Es por este fenymeno de proyectar una imagen 
virtual que suele denominarse al ocular como lente de 
proyecciyn. 
Como puede observarse en este ejemplo sobre- 
simplificado, la utilizaciyn del ocular da como resultado 
una imagen de mayor magnificaciyn que la lograda solo 
con el objetivo. Para calcular la magnificaciyn total 
lograda se utiliza la siguiente ecuaciyn: 
Mtotal = Mobjetivo x M ocular 
 
 
 
 
Donde vemos que cuando tenemos, por ejemplo, un 
objetivo 100X y utilizamos un ocular de 10X, la 
magnificaciyn de la imagen obtenida seri 1000 veces 
mayor al tamaxo original del objeto observado. 
El valor de magnificaciyn de los diferentes 
objetivos (y oculares) se encuentra inscripto en los 
mismos (Fig. 24-3). Cuando las imigenes son captadas 
por cimaras digitales, para conocer la magnificaciyn 
total, debe remplazarse la magnificaciyn del ocular por 
la magnificaciyn de la lentede acople que esté siendo 
utilizada. 
2.3. Contraste 
El contraste resulta un parimetro fundamental en 
el reconocimiento de estructuras por el observador. El 
ojo humano puede identificar un ltmite entre dos 
estructuras sólo si hay una diferencia de brillo (y ast de 
contraste) mayor al 2% y no gradual entre ellas. Es por 
esto que el correcto ajuste del contraste resulta 
fundamental en la observaciyn. Mientras que los dos 
parimetros anteriores (resoluciyn y magnificaciyn) 
Fig. 24-8. Ley de Snell. Esquema que muestra el cambio de velocidad (ltnea punteada) y direcciyn cuando una onda pasa de un medio 
de mayor a uno de menor tndice de refracciyn. Este cambio dependeri del ingulo de incidencia. 
 
 
Fig. 24-9. Ejemplo sobre simplificado de la marcha de rayos en un sistema yptico que consta de objetivo y ocular. El esppcimen situado a un distancia, mayor 
a la distancia focal (f) y menor a dos 2 veces a esta (2f), dari como resultado una imagen real invertida y magnificada, por efecto del objetivo (Imagen A). EL 
ocular por su parte generari a partir de esta Imagen A, una imagen invertida virtual, y de mayor tamaxo aun (Imagen B). 
Mtotal = Mobjetivo x Mocular
600x = 60x . 10x
• Magnificación
• Resolución 
• Contraste
 Microscopia Óptica
La parte fundamental del microscopio 
Magnificación Apertura Numerica
Resolución
8
CFI Plan Apochromat 
10XC Glyc
Compatible with a wide range of 
immersion media and clearing agents
In addition to water and immersion oil, this objective lens 
is compatible with a variety of tissue clearing agents. The 
lens also features chromatic aberration correction over a 
broad spectral range and is compatible with the Ti2 inverted 
microscope.
• NA: 0.50, WD: 5.50 mm (upright) / 2.00 mm (inverted)
• Chromatic aberration correction: from visible to IR
• High-transmittance Nano Crystal Coat
• Correction collar for spherical aberration correction
Cleared Tissue Imaging
Zebrafish larva
Plan Apo
60x/1.40 Oil
DIC
∞/ 0.17 WD 0.15
medio de inmersión
Código de color
de magnificación
Código de color
Correcciones óptica
Medio de inmersión
Magnificación
Diseño especializado
Distancia de Trabajo (WD)
Apertura numérica (NA)
Rosca
Lente
Espesor de cubreobjetos
Longitud del tubo
• Magnificación 
• Resolución
• Contraste
 Resolución
Resolución
Capacidad de un sistema óptico de 
diferenciar dos puntos como tales
John W. Strutt - 
Lord Rayleigh (1842-1919) 
El ojo humano pude distinguir dos puntos cuando el máximo de un punto coincide con el 
primer mínimo del otro. 
Criterio de Rayleigh
¿Cuál es la relación entre el 
disco de Airy y la resolución? 
Disco de Airy
Patrón de Airy
Disco de Airy
Patrón de Airy
97%
1,7%
97%
1,7%
¿Por qué el cielo es celeste? 
https://www.youtube.com/watch?
v=NqlxzUx7Z_E 
• Magnificación 
• Resolución
• Contraste
 Resolución
Resolución
Límite de Resolución 
(d0 ) = r
x
x
x
x
x
x
El ojo humano pude distinguir dos puntos cuando el 
máximo de un punto coincide con el primer mínimo 
Criterio de Rayleigh
• Magnificación 
• Resolución
• Contraste
 Microscopia
Resolución
Límite de Resolución 
(d0 )
d0
Capacidad de un sistema óptico de diferenciar dos puntos como tales
d= rairy = 0.61 𝜆 / sen 𝛳 
𝛳 
No es igual cuando luz es transmitida o 
reUlejada en la observación 
Ernst Karl Abbe 
(1840-1905) (Zeiss) 
Padre del microscopio 
moderno
≠
 Microscopia
Resolución
Límite de Resolución 
(d0 )
d0
Capacidad de un sistema óptico de diferenciar dos puntos como tales
d= rairy = 0.61 𝜆 / sen 𝛳 
𝛳 
d = 1,22 𝜆 /2.NA NA=(sen α) 𝑛 
luz transmitida
diferentes medios
do= 0,61 x 𝜆 
 AN
 Microscopia
Resolución
Límite de Resolución 
(d0 )
d0
Longitud onda
Apertura Numérica
do= 0,61 x 𝜆 
 AN
Capacidad de un sistema óptico de diferenciar dos puntos como tales
8
CFI Plan Apochromat 
10XC Glyc
Compatible with a wide range of 
immersion media and clearing agents
In addition to water and immersion oil, this objective lens 
is compatible with a variety of tissue clearing agents. The 
lens also features chromatic aberration correction over a 
broad spectral range and is compatible with the Ti2 inverted 
microscope.
• NA: 0.50, WD: 5.50 mm (upright) / 2.00 mm (inverted)
• Chromatic aberration correction: from visible to IR
• High-transmittance Nano Crystal Coat
• Correction collar for spherical aberration correction
Cleared Tissue Imaging
Zebrafish larva
Plan Apo
60x/1.40 Oil
DIC
∞/ 0.17 WD 0.15
medio de inmersión
Código de color
de magnificación
Código de color
Correcciones óptica
Medio de inmersión
Magnificación
Diseño especializado
Distancia de Trabajo (WD)
Apertura numérica (NA)
Rosca
Lente
Espesor de cubreobjetos
Longitud del tubo
Importancia del angulo
d0 = 0,61 . 𝜆 / AN
NA=(sen α) . 𝑛
α
NA=(sen 14,5º) . 1
α=14,5º
𝑛=1
𝜆=500 nm
NA=0,25
d0 = 0,61 . 500 nm / 0,25
d0 = 1220 nm
𝑛=1
α=64º
𝜆=500 nm
NA=(sen 64º) . 1
NA=0,9
d0 = 0,61 . 500 nm / 0,9 
d0 = 340 nm
Objetivo 1
α
Objetivo 2
Resolución y Apertura numérica
Importancia de 𝛂
La apertura numérica es una medida que nos indica la capacidad del objetivo de capturar la luz.
Cuanto más rayos pueda capturar el objetivo, mejor resolución tendrá.
NA=(sen α) . 𝑛
αα=14,5º
𝑛=1
𝜆=500 nm
𝑛=1
α=64º
𝜆=500 nm
Objetivo 1
α
Objetivo 2
Resolución y Apertura numérica
Importancia de 𝛂
La apertura numérica es una medida que nos indica la capacidad del objetivo de capturar la luz.
Cuanto más rayos pueda capturar el objetivo, mejor resolución tendrá.
Distancia de 
trabajo (WD)
Distancia de Trabajo
La refracción es el cambio de dirección y velocidad que experimenta una onda al pasar 
de un medio a otro con distinto índice refractivo.
Ley de Snell 
𝑛1 . sen 𝛳1 = 𝑛2 . sen 𝛳2
𝝷1
𝑛1
𝑛2
Interfase
𝝷2
𝝷1 𝝷1 𝝷2
𝝷2=90º
𝝷1 ángulo incidente
 haz incidente
haz resultante
𝝷2 ángulo refracción 
haz paralelo
haz reflejado
Propio de cada 𝜆
Resolución y Apertura numérica
Indice de refracciónImportancia de 𝑛 
NA=(senα) . 𝑛
 Importancia del 𝑛
Resolución y Apertura numérica
 Importancia del 𝑛
𝑛aire = 1.0 𝑛vidrio = 1.5 
𝑛vidrio = 1.5 
𝑛agua = 1.33 𝑛aceite ≅ 1.465
𝑛vidrio = 1.5 
NA=(senα) . 𝑛
Resolución y Apertura numérica
Importancia de 𝑛 
α=64º
𝜆=500 nm
d0 = 0,61 . 𝜆 / AN
NA=(sen 64º) . 1
NA=0,9
d0 = 0,61 . 500 nm / 0,9 
d0 = 340 nm
Objetivo 2
Lente Seca
NA=(sen 64º) . 1,515
NA=1,36
d0 = 0,61 . 500 nm / 1,36 
d0 = 224 nm
𝑛=1
Aire
𝑛=1,5
Vidrio 
Objetivo 2
Lente inmersión
𝑛=1,515
Aceite
 Importancia del 𝑛
X 9X
 Resolución vs Magnificación
Aumento no es sinónimo de Resolución
TV-LED 1080p (4K)
Resolución vs Magnificación
• Magnificación
• Resolución
• Contraste
Resolución no es sinónimode detección
 Resolución vs Detección
Resolución
Capacidad de un sistema óptico de diferenciar dos puntos como tales
Límite de Resolución
Resolución no es sinónimo de detección
D= 0.61x400nm/1.4 = 200nm
¿Puedo distinguir una estructura de menor tamaño? 
Puedo ver por debajo 
del límite de resolución
si emite luz 
¿Puedo ver una Proteína?
¿Puedo ver algo que es inferior a que mi capacidad visual?
Luciérnagas
 Resolución vs Detección
 Resolución vs Detección
Resolución
Capacidad de un sistema óptico de diferenciar dos puntos como tales
Límite de Resolución
D= 0.61x400nm/1.4 = 200nm
¿Puedo distinguir una estructura de menor tamaño? 
¿De qué tamaño se observa la estructura? 
Si la estructura es la emisora de luz, si. 
 Resolución vs Detección
Resolución vs Detección
20 nm
200 nm
Detecto 
no resuelvo
Visión no es absoluta sino 
comparativa 99Human Vision
© 2011 by Taylor & Francis Group, LLC
It is easy to confuse resolution with visibility. A star in the sky is essentially a point; there is no 
angular size, and even in a telescope most do not appear as a disk. A star is visible because of 
its contrast, appearing bright against the dark sky. Faint stars are not necessarily farther away 
or smaller, but are invisible to the naked eye because there isn’t enough contrast. Telescopes 
make them visible by collecting more light into a larger aperture. A better way to think about 
resolution is the ability to distinguish as separate two stars that are close together. The clas-
sic test for this has long been the star Mizar in the handle of the Big Dipper. In Van Gogh’s 
“Starry Night over the Rhone” each of the stars in this familiar constellation is shown as a 
single entity (Figure!2.14).
But a star chart shows that the second star in the handle is actually double. Alcor and Mizar 
are an optical double — two stars that appear close together but are at different distances and 
have no gravitational relationship to each other. They are separated by about 11.8 minutes of 
arc, and being able to detect the two as separate has been considered by many cultures from 
the American Indians to the desert dwellers of the near east as a test of good eyesight (as well 
 
Figure 2.12 Color patches used to test “just 
noticeable variations” in brightness or 
color, as described in the text.
(a) (b) 
Figure 2.13 Intensity differ-
ences superimposed on a 
varying background are 
visually undetectable:
 (a) original;
 (b) processed with an 
“un-sharp mask” filter 
to suppress the gradual 
changes and reveal the 
detail.
2% diferencia de brillo
Contraste
¿ Qué es el contraste ( ) ?
El contraste se define como la diferencia de intensidad (brillo) entre 
un punto de una imagen y su fondo. El ojo humano 2-5%
• Magnificación 
• Resolución 
• Contraste
¿Qué vemos?
Percepción de las Imágenes
La visión depende de la comparación local
NO ES POSIBLE DETERMINAR EL VALOR DE BRILLO ABSOLUTO
Comparativo - Expectativa
Contraste
• Magnificación 
• Resolución 
• Contraste
El tablero de Adelson
 “Checkershadow” 
Edward H. Adelson
 Contraste físico
¿Cómo lograr el contraste necesario?
Iluminación 
a) Voltímetro
Cambio Intensidad de 
la iluminación
 Contraste físico
¿Cómo lograr el contraste necesario?
Iluminación 
Disminución AN
a) Diafragma 
del condensador
Perdida de Resolución
nunca usar el diafragma 
para disminuir la intensidad 
de luz
Incremento Contraste 
Cambio de cono de 
iluminación
 Tipos de Microscopía Óptica
Microscopia Óptica
¿Cómo lograr el contraste necesario?
Tejidos
Células
[
dispersión refractiva
Con marcación Colorantes 
Luz blanca esta formada por diferentes longitudes de onda
 Tipos de Microscopía Óptica
Microscopia Óptica
¿Cómo lograr el contraste necesario?
Tejidos
Células
[
Con marcación Colorantes 
luz blanca
luz no 
absorbida
 Tipos de Microscopía Óptica
Microscopia Óptica
Tipos de Microscopia
Tejidos
Células
[
Sin marcación 
(contraste óptico)
Observación directa
Contraste de Fases
Campo Oscuro
Contraste de Fases 
interferencia diferencial 
(DIC - Nomarski)
Células vivas
Con marcación
Colorantes 
Contraste del espécimen Campo Oscuro
Sólo ingresa luz que sufre 
difracción y dispersión
Muy baja diferencia 
n del espécimen
Excelente contraste
Alta intensidad de luz
Artefactos
 Contraste Óptico
Sin marcación
Observación Claro
Campo Oscuro
Campo Oscuro
Contraste del espécimen Contraste de fases
Combina cambio de fase entre los 
diferentes haces de luz
Células vivas con poco espesor 
y homogéneas
Especímenes 
poco espesor e IR
Especimenes gruesos
Anillo de CF
- Atenuar luz no dispersada 
- Incremento en el cambio 
de fase en luz dispersada
Frits Zernike (1888-1966)
 Contraste Óptico
Contraste de Fases
Contraste de fases
Contraste del espécimen Contraste por interferencia diferencial (DIC)
Interferencia de haces de luz 
polarizada muy próximos 
Células vivas 
Especímenes 
Gruesos
Especimenes finos 
Polarizadores
Prismas de Wollastron
- Birefringente genera dos haces 
polarizados (opuestos) 
separados por 0,2 um 
- solo ingresa luz que haya 
- interactuado
George Nomarski (1919-1997)
 Contraste por interferencia diferencial (DIC) Contraste Óptico
DIC
Campo claro
 Time-lapse de microscopia DIC
 Tipos de Microscopía Óptica
Microscopia Óptica
Tipos de Microscopia
Tejidos
Células
[
Sin marcación
Observación directa
Contraste de Fases
Campo Oscuro
Contraste de Fases 
interferencia diferencial 
(DIC - Nomarski)
Con marcación
Colorantes Fluorescentes
Moléculas Orgánicas
Semiconductores
Sin marcación
Observación directa
Contraste de Fases
Campo Oscuro
Contraste de Fases 
interferencia diferencial 
(DIC - Nomarski)
Celulas vivas
¿Cómo lograr el máximo contraste?
El contraste se define como la diferencia de intensidad (brillo) entre un punto de una 
imagen y su fondo.
Fluorescencia
Contraste del espécimen
Gran contraste (epi-iluminación)
Observar la presencia de estructuras por 
debajo del límite de resolución
Contraste selectivo-Única molécula
 Fluorescencia
Fluorescencia
Fotoluminiscencia que se extingue al cesar la radiación que lo provoca (Vocabulario científico y técnico RACEFN)
Fotoluminiscencia que caracteriza a las sustancias que son capaces de absorber energía en forma de 
radiaciones electromagnéticas y luego emitir parte de esa energía en forma de radiación electromagnética 
de longitud de onda diferente.
Molecules 2012, 17 
 
4048
description of how these techniques are performed, what needs to be considered, and what 
practical advantages they can bring to cell biological research. 
Keywords: fluorescence microscopy; fluorescence; fluorochrome; techniques; confocal; 
multiphoton; anisotropy; FRET; homo-FRET; FRAP; FLIP; FLIM; FLAP 
 
1. Introduction 
FRAP, FLIP, FLAP, FRET, and FLIM are fluorescence microscopy techniques that in some way 
take advantage of particular aspects of the fluorescence process by which fluorochromes are excited 
and emit fluorescent light, are damaged during repetitive excitation, or undergo non-radiative decay 
prior to light emission. In order to understand the basic principles underpinning these advanced 
fluorescence techniques, first some general aspects of fluorescence and fluorescence microscopy are 
introduced before going into the technical details and practicalities of FRAP, FLIP, FLAP, FRET and 
FLIM. This article is not meant to be a comprehensive report on the aforementioned techniques, but 
rather to introduce these advanced fluorescence imaging techniques to a broad biological and 
bio(medical) research audience and give the reader some feeling for the field. The reader is referred to 
more specialized and comprehensive books and manuscripts for further reading throughout the text. 
1.1. Introduction to Fluorescence 
1.1.1. The Physical Phenomenon of Fluorescence 
Fluorescenceas a phenomenon is part of a larger family of related luminescent processes in which a 
susceptible substance absorbs light, only to reemit light (photons) from electronically excited states 
after a given time (Figure 1). Photoluminescent processes that are generated through excitation, 
whether this is via physical, mechanical, or chemical mechanisms, can generally be subdivided into 
fluorescence and phosphorescence. 
Figure 1. Fluorescence principle. Schematic representation of the fluorescence 
phenomenon in the classical Bohr model. Absorption of a light quantum (blue) causes an 
electron to move to a higher energy orbit. After residing in this “excited state” for a 
particular time, the 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
Tiempo- nanosegundos por los que puede considerarse instantáneo
Fluorescencia
Fosforescencia
Absorción - Desexitación no radiativa - Emisión
 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
FITC
DAPI Rodamina
Alexa 488
Cy5
 Quantum dot
Puntos Cuánticos
http://www.plasmachem.com
Molecules 2012, 17 
 
4058
Figure 6. (A) Molecular structure and localization of the chromophoric tripeptide in A. 
victoria wild-type GFP. Notice that the tripeptide is located centrally within the E-barrel. A 
vast number of genetically enhanced (denoted “E”, e.g., EGFP) and engineered FPs [27] 
have been created over the pasts decades. (B) Anatomy of a semiconductor quantum dot 
(QD), which derives its fluorescent properties from the bandgap between the inner core 
material and the capsule shell. QDs display size dependent fluorescent properties. 
(C) Excitation and emission spectra of A. victoria GFP (green lines) and examples of how 
the size influences the fluorescent properties of QDs. 
 
Molecules 2012, 17, 4047-4132
Mayor vida media que las moléculas orgánicas
 Fluoroforos
¿Cómo darle especificidad / selectividad?
F a) Fluorocromo con selectividad
F Anticuerpo c) Fluorocromo unido anticuerpo
F RNA/DNA b) Fluorocromo unido sonda hibridación 
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 
Gorlaeus Laboratory, Leiden State University, The Netherlands 
Department of Biology, Massachusetts Institute of Technology, Cambridge MA, USA 
* Departament d’Enginyeria Quimica, Universitat Politttcnica de Catalunya, Barcelona, Spain 
(Received February 15/March 24, 1994) - EJB 94 0220/2 
The molecular structure of the DNA A-tract dodecamer d(CGCAAATTTGCG) complexed with 
the drug Hoechst 33258 has been determined by X-ray diffraction analysis. The Hoechst molecule 
binds in the DNA minor groove covering the sequence AATTT of the central A-tract, with the 
piperazine group close to one of the GC regions. The drug molecule makes two three-centered 
hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and 0 2 atoms of the 
DNA bases. Although a high propeller twist is observed in the A-tract, only one unsymmetrical 
three-centered hydrogen bond is present in the DNA major groove. The structure is compared with 
other minor-groove-binding drug complexes and the influence of these drugs on DNA A-tracts is 
discussed. 
Hoechst 33258 [chemical name: 2’-(4-hydroxyphenyl)- 
5-(4-methyl-l-piperazinyl)-2,5’-bi-benzimidazole], netropsin 
and distamycin are DNA minor-groove-binding drugs with 
common structural features : (a) they are molecules contain- 
ing planar segments due to the presence of aromatics groups ; 
(b) they are non-symmetrical molecules ; (c) they are posi- 
tively charged and (d) they have an arc-like shape which 
allows them to follow the minor groove when interacting 
with the DNA double helix. Furthermore, these molecules 
have affinity for A+T-rich sequences over G+ C-containing 
sequences mostly due to steric factors (Burckhardt et al., 
1985). The presence of the N2 amino group in guanine pre- 
vents the binding of the drug in the DNA minor groove 
(Kopka et al., 1985), although Hoechst 33258 tolerates G-C 
pairs at the edge of the binding site (Harshman and Dervan, 
1985 ; Portugal and Waring, 1988). 
Hoechst 33258 is a synthetic N-methyl piperazine deriva- 
tive with two benzimidazole groups and one phenyl group 
(Fig. 1). It has antihelminthic activity (Lammler et al., 1971) 
and is commonly used as a fluorescent cytological stain for 
DNA (Hilwig and Gropp, 1972; Holmquist, 1975). Hoechst 
33258 binds preferentially to A+T-rich regions as deter- 
mined by fluorescence (Latt and Wohlleb, 1975 ; Holmquist, 
1975), footprinting (Harshman and Dervan, 1985) and ‘”1- 
DNA cleavage (Martin and Holmes, 1983; Murray and Mar- 
tin, 1988). 
There are several X-ray diffraction studies of this drug 
complexed with two different dodecamers. The complex 
Hoechst 33258-d(CGCGAATTCGCG) has been reported by 
Correspondence to M. Coll, Departament de Biologia Molecular 
i Cel.lular, Centre d’Investigaci6 i Desenvolupament-C. S. I. C., 
Jordi Girona 18, E-08034 Barcelona, Spain 
Abbreviation. Hoechst 33258, 2’-(4-hydroxyphenyl)-5-(4-meth- 
yl-1 -piperazinyl)-2,5’-bi-benzimidazole. 
Bz2 
OH 
Fig. 1. Atomic numbering scheme of the Hoechst 33258 molecule. 
The drug has four structural groups: a phenyl ring (Ph), two benzim- 
idazole rings (Bzl and Bz2) and a piperazine ring (Pip). The torsion 
angles between the four structural groups are indicated with arrows. 
The N1 and N3 atoms interact with the bases at the floor of the 
DNA minor groove. 
Pjura et al. (1987), Teng et al. (1988) and Quintana et al. 
(1991). The different analyses show some discrepancies in 
the positioning of the drug along the minor groove which 
might be due to different crystallization conditions or dif- 
 Fluoroforos - especificidad/selectividad
Fluorocromo unido a moléculas 
Faloidina-(FITC)
Favale et al JLR (2015)
Toxina
F-actina 
Fluorescente
verde 
F Afinidad
 Fluoroforos - especificidad/selectividad
Fluorocromo unido sonda 
FISH 
(Fluorescence In-Situ Hybridization)
F RNA/DNA
T. Liehr, H. et al Multicolor FISH probe sets and their applications 
Histol Histopathol (2004) 19: 229-237 
This basic mFISH probe set (Fig. 1) has been
modified either by molecular changes in the probes
themselves or by addition of supplementary probes. The
so-called IPM-FISH (= IRS-PCR multiplex FISH)
method uses whole chromosome painting probes which
are modified by an interspersed polymerase chain
reaction (IRS), which leads to a 24-color-FISH painting
plus an R-band-like pattern (Aurich-Costa et al., 2001).
For special questions other probes were added to the
basic 24-color-FISH probe set, like single copy probes
(e.g. probe for human papillomavirus (Szuhai et al.,
2000, 2001; Brink et al., 2002) or subtelomeric probes
(Tosi et al., 1999)), chromosome-region-specific probes
(e.g. a probe for the short arm of all acrocentric
chromosomes (Mrasek et al., 2001) – Fig. 1) or
chromosome-arm-specific probes for all human
chromosomes (42-color-FISH (Wiegant et al., 2000;
Karhu et al., 2001; Brink et al., 2002; Liehr and
Claussen, 2002).
mFISHbanding probe sets
FISH banding probe sets are defined as “any kind of
FISH technique, which provides the possibility to
simultaneously characterize several chromosomal
subregions smaller than a chromosome arm - excluding
the short arms of the acrocentric chromosomes; FISH
banding methods fitting that definition may have quite
different characteristics, but share the ability to produce
a DNA-specific chromosomal banding” (Liehr et al.,
2002). In the following paragraphs the available mFISH
banding probe sets are listed according to their quality of
resolution.
1. The cross-species color banding (Rx-FISH) or
Harlequin-FISH probe set (Fig. 2) provides the lowest
resolution of 80-90 bands per haploid human karyotype
(Müller and Wienberg, 2000). The probe set consists of
flow-sorted gibbon chromosomes, which are labeled
with three different fluorochromes (Müller et al., 1998).
A set of 110 human-hamster somatic cell hybrids, split
into two pools and labeled with two fluorochromes
(Müller et al., 1997), leads, when hybridized to human
chromosomes, to about 100 “bars” on each chromosome.
This pattern has been called ‘somatic cell hybrid-based
chromosome bar code’. A combination the Rx-FISH
probe set with the 110 somatic cell hybrid probes results
in 160 chromosome-region-specific DNA-mediated
bands in human karyotypes (Müller and Wienberg, 2000;
Müller et al., 2002).
2. An approach called SCAN (= spectral color
banding) has been described exemplarily for one
chromosome up to present. 8 microdissection libraries
were created along chromosome 10 with the aim of
obtaining a banding pattern similar to the GTG-banding
at the 300 band level (Kakazu et al., 2001).
3. A chromosome can be characterized as well by a
specific signal pattern produced by region-specific YAC
(= yeast artificial chromosomes) clones. The first
attempts to label each chromosome by subregional DNA
probes in different colors were performed by the groups
of David Ward (Lichter et al., 1990) and Thomas Cremer
(Lengauer et al., 1993). A YAC-based chromosome bar
code has been especially created for chromosome 12 but
not for the entire human karyotype yet (for review see
(Liehr and Claussen, 2002, 2002a)). A resolution of up
to 400 bands can be achieved, depending on the number
of applied probes.
4. The aforementioned IPM-FISH approach (Aurich-
Costa et al., 2001) can be categorized as an mFISH
banding probe set, as well. A resolution of about 400
bands per haploid karyotype can be attained, dependent
on the chromosome quality.
5. The high-resolution multicolor-banding (MCB)
technique, based on overlapping microdissection
libraries producing fluorescence profiles along the
human chromosomes was described first on the example
230
Multicolor FISH probe sets
Fig. 1. 25-color FISH karyogram of a normal female metaphase (Mrasek
et al., 2001). Like in M-FISH or SKY each chromosome is labeled in a
different (pseudo-)color. Addit ionally to the 24 human whole
chromosome painting probes (as it is a female no Y-chromosome is
present), a probe specific for all short arms of human acrocentric
chromosomes, i.e. #13, #14, #15, #21, and #22 (marked by
arrowheads), is included. The probe is microdissection-derived and has
been called midi54 – in the legend for the pseudocolors for each
individual chromosome the 25th color for midi54 is abbreviated as “M”.
Fig. 2. Rx-FISH performed on a normal female metaphase.
Chromosomes can be distinguished based on three fluorochromes and
~90 bands per haploid karyotype. However, e.g. chromosomes 21, 22 or
X are not divided into subbands in that assay. 
 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 
 Tipos de Microscopía Óptica
Microscopia Óptica
Tipos de Microscopia
Sin marcación
Observación directa
Contraste de Fases
Campo Oscuro
Contraste de Fases 
interferencial 
(DIC - Nomarski)
Fluorescentes
Moléculas Orgánicas
Semiconductores
Con marcación
Colorantes 
Enzimas
Microscopia Electrónica
 Microscopia
Tipos de Microscopía
Microscopía Óptica
Microscopía Electrónica ✔
Microscopio electrónicoAumentando la resolución
Microscopia Electrónica
Microscopio electrónicoAumentando la resolución
d0 = 0,61 . 𝜆 / AN
aplica para cualquier 
onda electromagnética
380-750 nm do= 200 nmMicroscopia Óptica
0,002 nmMicroscopia Electrónica doteorico= 0,001 nm doreal= 0,1 nm
 Microscopia
Tipos de Microscopía
Microscopía Óptica
Microscopía Electrónica ✔
doreal= 0,1 nm
Microscopio electrónicoAumentando la resolución
Organelas
Microscopia Óptica Microscopia Electrónica
200 nm 0,1 nm
VACIO
Microscopio Electrónico de Transmisión 
(MET o TEM )
Muestras 
Preparación de las muestras
Contrastar (Os, Pb y U) - electrodenso
e- e-
Fijar (GA y TO4Os) 
Deshidratar 
Secciones 50-100 nm
158 CHAPTER 4 t Culturing and Visualizing Cells
coated with a thin film of plastic and carbon. The sample 
is then bathed in a solution of a heavy metal, such as ura-
nyl acetate, and excess solution is removed (Figure 4-29b). 
As a result of this procedure, the uranyl acetate coats the 
grid, but is excluded from the regions where the sample 
has adhered. When we view the sample in the TEM, we see 
where the stain has been excluded, so the sample is said to 
be negatively stained. Because the stain can precisely reveal 
the topology of the sample, a high-resolution image can be 
obtained (Figure 4-29c).
Samples can also be prepared by metal shadowing 
(Figure 4-30). In this technique, the sample is absorbed to 
a s mall piece of mica, then coated with a thin film of plati-
num by evaporation of the metal, then dissolved with acid 
or bleach, leaving the platinum coating (known as a replica). 
The platinum coating can be generated from a fixed angle 
or at a low angle as the sample is rotated, in which case it is 
called low-angle rotary shadowing. When the replica is trans-
ferred to a grid and examined in the TEM, it provides infor-
mation about the three-dimensional topology of the sample.
FIGURE 4!30 Metal shadowing makes surface details on very 
small objects visible by transmission electron microscopy. (a) 
The sample is spread on a mica surface and then dried in a vacuum 
evaporator (step 1 ). The sample grid is coated with a thin film of a 
heavy metal, such as platinum or gold, evaporated from an electrically 
heated metal filament (step 2 ). To stabilize the replica, the specimen is 
then coated with a carbon film evaporated from an overhead elec-
trode (step 3 ). The biological material is then dissolved by acid and 
bleach (step 4 ), and the remaining metal replica is viewed in a TEM. 
In electron micrographs of such preparations, the carbon-coated!areas 
appear light—the reverse of micrographs of simple metal-stained 
preparations, in which the areas of heaviest metal staining appear 
the!darkest. (b) A platinum-shadowed replica of poliovirus particles. 
[Part (b) Science Source]
Sample Mica surface
Metal replica
Evaporated carbon
Evaporated platinum
Carbon film
Metal replica ready
for visualization
1
2
3
4
(a) (b)
0.5 µm
Cells and Tissues Are Cut into Thin Sections 
for Viewing by Electron Microscopy
Single cells and pieces of tissue are too thick to be viewed 
directly in the standard transmission electron microscope. To 
overcome this problem, methods were developed to prepare 
and cut thin sections of cells and tissues. When these sections 
were examined in the electron microscope, the organization, 
beauty, and complexity of the cell interior was revealed and 
led to a revolution in cell biology—for the first time, new 
organelles and the first glimpses of the cytoskeleton were seen.
To prepare thin sections, it is necessary to chemically fix 
the sample, dehydrate it, impregnateit with a liquid plastic 
that hardens (similar to Plexiglas), and then cut sections of 
about 5 to 100 nm in thickness. For structures to be seen, the 
sample has to be stained with heavy metals such as uranium 
and lead salts, which can be done either before embedding 
in the plastic or after sections are cut. Examples of cells and 
tissues viewed by thin-section electron microscopy appear 
here and throughout this book (Figure 4-31). It is important 
Réplica (sombreado)
Crioelectrónico (cryo-EM) 
(Congelado alta presión-etano)
Nobel 2017 
Dubochet, Frank, Henderson
 Microscopia electrónica
18 CHAPTER 1 t Molecules, Cells, and Model Organisms
storage vacuoles are found in green algae and in many mi-
croorganisms such as fungi.
Mitochondria Are the Principal Sites 
of ATP Production in Aerobic Cells
Most eukaryotic cells contain many mitochondria 
(Figure 1-20), which occupy up to 25 percent of the volume 
of the cytoplasm. These complex organelles, which are the 
main sites of ATP production during aerobic metabolism, 
are generally exceeded in size only by the nucleus, vacuoles, 
and chloroplasts. The two membranes that bound a mito-
chondrion differ in composition and function. The outer 
mitochondrial membrane contains proteins that allow many 
molecules to move from the cytosol to the intermembrane 
space between the inner and outer membrane. The inner 
mitochondrial membrane, which is much less permeable, is 
about 20 percent lipid and 80 percent protein—a proportion 
of protein that is higher than those in other cellular mem-
branes. The surface area of the inner membrane is greatly 
increased by a large number of infoldings, or cristae, that 
protrude into the matrix, or central aqueous space.
In non-photosynthetic cells, the principal fuels for ATP 
synthesis are fatty acids and glucose. The complete aerobic 
degradation of 1 molecule of glucose to carbon dioxide and 
water is coupled to the synthesis of as many as 30 molecules 
of ATP from ADP and inorganic phosphate (see Figure 1-6). 
In eukaryotic cells, the initial stages of glucose degradation 
take place in the cytosol, where 2 ATP molecules per glucose 
molecule are generated. The terminal stages of oxidation and 
Outer
membraneCristaeInner membrane
Intermembrane
space Matrix
granules
Matrix
3 Rm
FIGURE 1!20 Electron micrograph of a mitochondrion in a pan-
creas cell. The smooth outer membrane forms the outside boundary 
of the mitochondrion. The inner membrane is distinct from the outer 
membrane and is highly invaginated to form sheets and tubes called 
cristae; ATP is produced by proteins embedded in the membranes of 
the cristae. The aqueous space between the inner and outer mem-
branes (the intermembrane space) and the space inside the inner 
membrane (the matrix) each contain specific proteins important for 
the metabolism of sugars, lipids, and other molecules. [Keith R. Porter/
Science Source.]
ATP synthesis are carried out by enzymes in the mitochon-
drial matrix and inner membrane (see Chapter 12); as many 
as 28 ATP molecules per glucose molecule are generated in 
mitochondria. Similarly, virtually all the ATP formed in the 
oxidation of fatty acids to carbon dioxide is generated in 
mitochondria. Thus mitochondria can be regarded as the 
“power plants” of the cell.
Mitochondria contain small DNA molecules that encode 
a small number of mitochondrial proteins; the majority of 
mitochondrial proteins are encoded by nuclear DNA. As dis-
cussed in Chapter 12, the popular endosymbiont hypothesis 
postulates that mitochondria originated by endocytosis of 
an ancient bacterium by the precursor of a eukaryotic cell; 
the bacterial plasma membrane evolved to become the inner 
mitochondrial membrane.
Chloroplasts Contain Internal Compartments 
in Which Photosynthesis Takes Place
Except for vacuoles, chloroplasts are the largest and the 
most characteristic organelles in the cells of plants and green 
algae (see Figure 1-19). The endosymbiont hypothesis (see 
Chapter 12) posits that these organelles originated by endo-
cytosis of a primitive photosynthetic bacterium. Chloroplasts 
can be as long as 10 !m and are typically 0.5–2 !m thick, 
but they vary in size and shape in different cells, especially 
among the algae. In addition to the inner and outer mem-
branes that bound a chloroplast, this organelle also contains 
an extensive internal system of interconnected membrane-
limited vesicles called thylakoids, which are flattened to 
form disks. Thylakoids often form stacks called grana and 
are embedded in an aqueous matrix termed the stroma. The 
thylakoid membranes contain green pigments (chlorophylls) 
and other pigments that absorb light, as well as enzymes 
that generate ATP during photosynthesis. Some of the ATP 
is used to convert carbon dioxide into three-carbon interme-
diates by enzymes located in the stroma; the intermediates 
are then exported to the cytosol and converted into sugars.
The molecular mechanisms by which ATP is formed in 
mitochondria and chloroplasts are very similar, as explained 
in Chapter 12. Besides being surrounded by two membranes, 
chloroplasts and mitochondria have other features in com-
mon: both often migrate from place to place within cells, 
and both contain their own DNA, which encodes some of 
the key organelle proteins (see Chapter 12). The proteins 
encoded by mitochondrial or chloroplast DNA are synthe-
sized on ribosomes within the organelles. However, most of 
the proteins in each organelle are encoded in nuclear DNA 
and are synthesized in the cytosol; these proteins are then 
 incorporated into the organelles by processes described in 
Chapter 13.
All Eukaryotic Cells Use a Similar Cycle 
to Regulate Their Division
Unicellular eukaryotes, animals, and plants all use essentially 
the same cell cycle, the series of events that prepares a cell to 
Preparación de las muestras
 Microscopia electrónica
Tipos de Microscopia eletrónica
4.3 Electron Microscopy: High-Resolution!Imaging 157
optical lenses. In the transmission electron microscope (TEM), 
electrons are emitted from a filament and accelerated in an 
electric field (Figure 4-28, left). A condenser lens focuses the 
electron beam onto the sample; objective and projector lenses 
focus the electrons that pass through the specimen and project 
them onto a viewing screen or other detector. Because atoms 
in air absorb electrons, the entire tube between the electron 
source and the detector is maintained under an ultrahigh 
vacuum. Thus living material cannot be imaged by electron 
microscopy.
In this section, we describe various approaches to viewing 
biological material by electron microscopy. The most widely 
used instrument is the transmission electron microscope, 
but also in common use is the scanning electron microscope 
(SEM), which provides complementary information, as we 
discuss at the end of this section (Figure 4-28, right).
FIGURE 4!28 In electron microscopy, images are formed from 
electrons that pass through a specimen or are scattered from a 
metal-coated specimen. In a transmission electron microscope (TEM, 
left), electrons are extracted from a heated filament, accelerated by an 
electric field, and focused on the specimen by a magnetic condenser 
lens. Electrons that pass through the specimen are focused by a series 
of magnetic objective and projector lenses to form a magnified image 
of the specimen on a detector, which may be a fluorescent viewing 
screen, a photographic film, or a charged-couple-device (CCD) camera. 
In a scanning electron microscope (SEM), electrons are focused by 
condenser and objective lenses on a metal-coated specimen. Scanning 
coils move the beam across the specimen, and electrons scattered 
from the metal are collected by a photomultiplier tube detector. In 
both types of microscopes, because electrons are easily scattered by air 
molecules, the entire column is maintained at a very high vacuum.
Tungsten filament
(cathode) 
Anode
SEMTEM
Beamof electrons
Scanning
coils
Condenser lens
Specimen
Specimen
Electromagnetic
objective lens
Projector lens
Detector
Single Molecules or Structures Can Be Imaged 
Using a Negative Stain or Metal Shadowing
It is common in biology to explore the detailed shapes of 
single macromolecules, such as proteins or nucleic acids, 
or of structures, such as viruses and the filaments that 
make up the cytoskeleton. It is relatively easy to view these 
objects in the transmission electron microscope, provided 
they are stained with a heavy metal that scatters the inci-
dent electrons. To prepare a sample, it is first absorbed to 
a 3-mm electron microscope grid (Figure 4-29a), which is 
FIGURE 4!29 Transmission electron microscopy of negatively 
stained samples reveals fine features. (a) Samples for transmission 
electron microscopy (TEM) are usually mounted on a small copper or 
gold grid. The grid is usually covered with a very thin film of plastic 
and carbon to which a sample can adhere. (b) The specimen is then 
incubated in a heavy metal, such as uranyl acetate, and excess stain is 
removed. (c) The sample excludes the stain, so when it is observed in 
the TEM, it is seen in negative outline. The example in (c) is a negative 
stain of rotaviruses. [Part (c) ISM/Phototake.]
(b)(a)
Add
sample
3 mm Stain sample
with heavy
metal
(c)
100 nm
Filamento Tungsteno
Anodo
Condensador
Haz de electrones
Deflector
Microscopio Electrónico de Transmisión 
(MET o TEM)
Microscopio Electrónico de Barrido 
(MEB o SEM )
Lente 
objetivo 
electromagnéticas
Lente 
proyección 
electromagnéticas
Detector
espécimen
espécimen
0,1 nm 10 nm
Microscopio electrónicoAumentando la resolución
1.5 Metazoan Structure, Differentiation, and Model Organisms 25
studies have shown that many of them are essential for the 
formation and function of specific tissues and organs. Thus 
many of the organisms listed in Table 1-2 are used to study 
the roles of these conserved proteins in cell development and 
function.
While the human and mouse genomes encode about the 
same number of proteins as those of the roundworm Cae-
norhabditis elegans, frogs, and fish, mammalian cells con-
tain about 30 times the DNA of a roundworm and two to 
three times the DNA of frogs and fish. Only about 10 per-
cent of human DNA encodes proteins. We know now that 
much of the remaining 90 percent has important functions. 
Many DNA segments bind proteins that regulate expression 
of nearby genes, allowing each mammalian gene to make 
the precise amount of mRNA and protein needed in each of 
many types of cells.
Other segments of DNA are used to synthesize thousands 
of RNA molecules whose function in regulating gene expres-
sion is only now being uncovered. As an example, hundreds 
of different micro-RNAs, 20 to 25 nucleotides long, are 
abundant in metazoan cells, where they bind to and repress 
the activity of target mRNAs. These small RNAs may in-
directly regulate the activity of most or all genes, either by 
inhibiting the ability of mRNAs to be translated into pro-
teins or by triggering the degradation of target mRNAs (see 
Chapter 10).
Some of this non-protein-coding DNA probably regu-
lates expression of genes that make us uniquely human. 
Indeed, fish and humans have about the same number of 
protein-coding genes—about 20,000—yet as noted above, 
the human genome is over twice the size of that in fish (see 
Table 1-2). The human brain can perform complex mental 
processes such as reading and writing a textbook. Somehow 
these 20,000 human genes are exquisitely regulated such 
that humans produce a brain with about 100,000,000,000 
neurons, which communicate with one another at about 
100,000,000,000,000 interaction sites termed synapses.
Genomics—the study of the entire DNA sequences of or-
ganisms—has shown us how close humans really are to our 
nearest relatives, the great apes (Figure 1-26). Human DNA 
is 99 percent identical in sequence to that of chimpanzees 
and bonobos; the 1 percent difference is about 3,000,000 
base pairs, but it somehow explains the obvious differences 
between our species, such as the evolution of human brains 
during the past 5,000,000 years since we last shared a com-
mon ancestor.
Genomics coupled with paleontological findings indi-
cates that humans and mice descended from a common 
mammalian ancestor that probably lived about 75 million 
years ago. Nonetheless, both organisms contain about the 
same number of genes, and about 99 percent of mouse 
protein-coding genes have homologs in humans, and vice 
versa. Over 90 percent of mouse and human genomes can 
be partitioned into regions of synteny—that is, DNA seg-
ments that have the same order of unique DNA sequences 
and genes along a segment of a chromosome. This observa-
tion suggests that much of the gene order in the most recent 
common ancestor of humans and mice has been conserved 
in both species (Figure 1-27). Of course, mice are not peo-
ple; relative to humans, mice have expanded families of 
genes related to immunity, reproduction, and olfaction, 
probably reflecting the differences between the human and 
mouse lifestyles.
It’s not only human evolution that interests us! Polar 
bears live in the Arctic and eat a high-fat diet, mostly com-
posed of seals. Recent genome sequencing allowed research-
ers to conclude that the most recent common ancestor of 
polar bears and their brown bear relatives, which live in tem-
perate climates, was present about 500,000 years—or only 
about 20,000 bear generations—ago. But during that rather 
short evolutionary period the polar bear genome acquired 
changes in many genes regulating cardiovascular function, 
fat metabolism, and heart development, allowing it to con-
sume a diet very rich in fats.
Embryonic Development Uses a Conserved Set 
of Master Transcription Factors
The astute reader will note a paradox in the previous discus-
sion: if indeed most human protein-coding genes are shared 
with apes and mice, and many with flies and worms, how 
is it that these organisms look and function so differently? 
FIGURE 1!25 All organs are organized arrangements of vari-
ous tissues, as illustrated in this cross section of a small artery 
(arteriole). Blood flows through the vessel lumen, which is lined by 
a thin sheet of endothelial cells forming the endothelium and by the 
underlying basal lamina. This tissue adheres to the overlying layer of 
smooth muscle tissue; contraction of the muscle layer controls blood 
flow through the vessel. A fibrillar layer of connective tissue surrounds 
the vessel and connects it to other tissues. [SPL/Science Source.]
Connective
tissue
Smooth
muscle
Lumen
Endothelium
4.4 Isolation of Cell Organelles 161
into a three-dimensional reconstruction termed a tomogram 
(Figure 4-34c, d). A disadvantage of cryoelectron tomography 
is that the samples must be relatively thin, about 200 nm; this 
is much thinner than the samples (200!!m thick) that can be 
studied by confocal light microscopy.
Scanning Electron Microscopy of Metal-Coated 
Specimens Reveals Surface Features
Scanning electron microscopy (SEM) allows investigators 
to view the surfaces of unsectioned metal-coated specimens. 
An intense electron beam inside the microscope scans rap-
idly over the sample. Molecules in the coating are excited 
and release secondary electrons that are focused onto a 
scintillation detector; the resulting signal is displayed on 
a cathode-ray tube much like a conventional television 
(see Figure 4-28, right). The resulting scanning electron 
micrograph has a three-dimensional appearance because the 
number of secondary electrons produced by any one point 
on the sample depends on the angle of the electron beam in 
relation to the surface (Figure 4-35). The resolving power of 
scanning electron microscopes, which is limited by the thick-
ness of the metal coating, is only about 10 nm, much less 
than that of transmission instruments.
4.4 Isolation of Cell Organelles
Theexamination of cells by light and electron microscopy 
led to the appreciation that eukaryotic cells contain a 
common set of organelles, introduced in Chapter 1 (see 
Figure 1-12a). However, observing organelles and docu-
menting their detailed structure by microscopy does not 
clearly reveal the roles they play and how they work. For 
this, it is necessary to isolate organelles in their native state 
and identify and dissect the function of each component. For 
this reason, methods to isolate and characterize organelles 
were developed in parallel with advances in microscopy. 
Lysosomes, for example, are organelles in which bio-
logical molecules are degraded, as described in Chapter 1. 
 Lysosomes had been seen by microscopy, but their function 
was discovered only after a method was developed to isolate 
Cilia
Goblet
cell
Basal
lamina
Epithelial
cells
10�!m
FIGURE 4!35 Scanning electron 
microscopy (SEM) produces a three-
dimensional image of the surface of 
an unsectioned specimen. Seen here 
is an SEM image of cells of the trachea. 
In the middle is a goblet cell, which 
secretes mucus. On either side of the 
goblet cell are epithelial cells with 
abundant cilia on their apical surfaces. 
[Steve Gschmeissner/Science Source.]
r Simple specimens, such as proteins or viruses, can be nega-
tively stained or shadowed with heavy metals for examina-
tion in a transmission electron microscope (TEM).
r Thicker sections generally must be fixed, dehydrated, em-
bedded in plastic, sectioned, and then stained with electron-
dense heavy metals before viewing by TEM.
r Specific proteins can be localized by TEM by employing 
specific antibodies associated with a heavy metal marker, 
such as small gold particles.
r Cryoelectron microscopy allows examination of hydrated, 
unfixed, and unstained biological specimens in the TEM by 
maintaining them at very low temperatures.
r Scanning electron microscopy (SEM) of metal-shadowed 
material reveals the surface features of specimens.
KEY CONCEPTS OF SECTION 4.3
Electron Microscopy: High-Resolution 
 Imaging
r Electron microscopy provides very high-resolution images 
because of the short wavelength of the high-energy electrons 
used to image the sample.
 561
being plunged into a coolant. A special sample holder keeps this hydrated speci-
men at –160°C in the vacuum of the microscope, where it can be viewed directly 
without !xation, staining, or drying. Unlike negative staining, in which what we 
see is the envelope of stain exclusion around the particle, hydrated cryoelectron 
microscopy produces an image from the macromolecular structure itself. How-
ever, the contrast in this image is very low, and to extract the maximum amount 
of structural information, special image-processing techniques must be used, as 
we describe next.
Multiple Images Can Be Combined to Increase Resolution
As we saw earlier (p. 532), noise is important in light microscopy at low light levels, 
but it is a particularly severe problem for electron microscopy of unstained mac-
romolecules. A protein molecule can tolerate a dose of only a few tens of electrons 
per square nanometer without damage, and this dose is orders of magnitude 
below what is needed to de!ne an image at atomic resolution.
"e solution is to obtain images of many identical molecules—perhaps tens 
of thousands of individual images—and combine them to produce an averaged 
image, revealing structural details that are hidden by the noise in the original 
images. "is procedure is called single-particle reconstruction. Before com-
bining all the individual images, however, they must be aligned with each other. 
Sometimes it is possible to induce proteins and complexes to form crystalline 
arrays, in which each molecule is held in the same orientation in a regular lattice. 
In this case, the alignment problem is easily solved, and several protein structures 
have been determined at atomic resolution by this type of electron crystallogra-
phy. In principle, however, crystalline arrays are not absolutely required. With the 
help of a computer, the digital images of randomly distributed and unaligned mol-
ecules can be processed and combined to yield high-resolution reconstructions 
(see Movie 13.1). Although structures that have some intrinsic symmetry make 
the task of alignment easier and more accurate, this technique has also been used 
for objects like ribosomes, with no symmetry. Figure 9–54 shows the structure of 
LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE
Figure 9–52 The nuclear pore. Rapidly 
frozen nuclear envelopes were imaged in 
a high-resolution SEM, equipped with a 
field emission gun as the electron source. 
These views of each side of a nuclear pore 
represent the limit of resolution of the SEM 
(compare with Figure 12–8). (Courtesy of 
Martin Goldberg and Terry!Allen.)
CYTOSOL
nuclear
pore
NUCLEUS
50 nm
MBoC6 m9.51/9.52
100 nm
MBoC6 m9.54/9.53
Figure 9–53 Negatively stained actin 
filaments. In this transmission electron 
micrograph, each filament is about 8 nm in 
diameter and is seen, on close inspection, 
to be composed of a helical chain of 
globular actin molecules. (Courtesy of 
Roger Craig.)
WHAT WE DON’T KNOW
• We know in detail about many cell 
processes, such as DNA replication 
and transcription and RNA translation, 
but will we ever be able to visualize 
such rapid molecular processes in 
action in cells? 
• Will we ever be able to image 
intracellular structures at the resolution 
of the electron microscope in living 
cells?
• How can we improve crystallization 
and single-particle cryoelectron 
microscopy techniques to obtain high-
resolution structures of all important 
membrane channels and transporters? 
What new concepts might these 
structures reveal?
 Tipos de Microscopía Óptica
Tipos de Microscopia
Microscopia Óptica
Sin marcación
Observación directa
Contraste de Fases
Campo Oscuro
Contraste de Fases 
interferencial 
(DIC - Nomarski)
Fluorescentes
Moléculas Orgánicas
Semiconductores
Proteínas Celulas vivas
Con marcación
Colorantes 
Enzimas
Microscopia Electrónica
• Magnificación 
• Resolución 
• Contraste
Microscopio Electrónico de Transmisión 
(MET o TEM)
Microscopio Electrónico de Barrido 
(MEB o SEM )
Muchas	Gracias