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Fluorescence Light Microscopy

for Cell Biology

Structure within a cell

Locations of specific molecules within a cell

Traditional questions that light microscopy has addressed:

Recent advances now allow these

questions to be asked in live cells!

Why use light microscopy?

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Determine diffusion constants and binding affinities of

a molecule at a specific site within a cell.

Determine whether two molecules are interacting at a

specific time and place within a cell.

Non-traditional “biophysical” questions that light

microscopy can now address:

Much more information than just structure!

Why use light microscopy?

Outline of topics

Conventional fluorescence microscopy

Confocal microscopy

Deconvolution microscopy

Two-photon microscopy

Microscopes:

Imaging techniques:

3D time-lapse

FRAP

FRET

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Web sites for more

information and tutorials

http://microscopy.fsu.edu/primer/index.html

Molecular Expressions Optical Microscopy Primer

http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html

Olympus microscopy resource center

www.molecularprobes.com

Molecular Probes

Basic operating principles of a

light microscope

objective lens

specimenfocus

eyepiece

condenser

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Compound microscope design yields

magnification and resolution

Magnification

5x, 10x, 20x, 25x, 40x, 60x, 100x typically available

for microscope objectives

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Resolution is determined in part by

the imaging medium

n = refractive index of medium:

n=1.0 air; n=1.3 water; n=1.5 oil.

(for glass n=1.5)

The more light collected, the more complete is the

image, and so resolution improves.

AIR OIL

Resolution quantified by

numerical aperture (NA)

sinNAn

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What’s the resolution limit

of light microscopy?

Rayleigh criterion: d = /(2)(NA)s N nn

d/ n

d500/ .5 700. 7nmmμ]

d

Beware of empty

magnification

High NA Low NA

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Inverted Research Microscope

new

detector

old

detector

original

detector

Detector

Computer

Microscope

Microscopist

(Who needs a microscopist?)

Computer controlled

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Fluorescence Microscopy

Predominant mode of light microscopy today

Provides molecular specificity

Yields high signal to background

What is fluorescence?

Absorption of a photon with emission of

longer wavelength photon

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higher energy

(shorter wavelengths)

lower energy

(longer wavelengths)

Fluorescence is absorption of a higher energy

photon with emission of a lower energy photon

Typical spectral curves for a fluorescent

molecule used in microscopy

How do we specifically excite the molecule, and

then specifically detect its fluorescence?

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Specificity provided by filters

Filters plus a dichroic mirror

Fluorescence Microscopy

Exciter filter

Objective

SpecimenSpecimen

Dichroic mirror

Emission filter

Filters plus a dichroic mirror

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Components combined in a

small filter cube

Fluorescence Inverted Research Microscope

excitation

filters,

dichroic

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What can we see by

fluorescence microscopy?

Probes for specific biomolecules

Probes for genes

Probes for ions

Probes for specific biomolecules

Fluorescent antibodies (immunofluorescence)

Fluorescent biomarkers

cellular molecule

antibody or other binding molecule

conjugated fluorescent dye

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Chromosome axis with topo II Microtubules in an endothelial cell

Cell walls in plant cells Histone proteins (green), EGF receptor (red)

Examples of immunofluorescence

Examples of fluorescent biomarkers

Metaphase chromosomes

Nucleic acid (yellow)Apoptotic cell: Lectin

(green), nucleic acid (red)Actin filaments

(tubeworm)

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Fluorescent probes for cellular organelles

Endoplasmic reticulum Golgi

Mitochondria Lysosomes (red), Nucleus (blue)

Fluorescent probes for small

signaling molecules

calcium

(pollen tube)phosphatidyl inositol

(fibroblasts)

cAMP

(fibroblasts)

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Conclusion:

An assortment of fluorescent probes enables detection

of a variety of cellular structures and organelles.

A more limited assortment of fluorescent probes

permits detection of small signaling molecules that

regulate cell processes.

But cells are 3D!!!

How do we get a 3D image of a cell?

focus

Easy, change the focus.

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This is called optical

sectioning microscopy

Acquire a series of focal plane images that

span the depth of the cell or object of interest.

Example: collecting a 3D image ofa tiny fluorescent bead (~0.2 μm)

z

focal planes

+4 μm

+3 μm

+2 μm

+1 μm

+0 μm

-1 μm-2 μm

-3 μm

-4 μm

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3D image stack from the small

fluorescent bead

focal planes

(xy images)

xz section

The 3D bead image viewed

from the side

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But why didn’t we get -

?

xz section

xy sections

Because the lens does not collect all of

the light emitted by the specimen

Therefore the image formed is imperfect.

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So in 3D the image of a point

source always looks like this:

out-of-focus light

Out-of-focus light creates

blur in a 3D image

Image Formation

Selected focal plane image

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in focus 1 μm out-of-focus

Image of many real point sources

A real specimen is composed of many

more such point sources

3D microscopy methods to reduce

out-of-focus light

Confocal microscopy

Two-photon microscopy

Deconvolution microscopy

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Conventional fluorescence microscopy

excites the whole specimen and collects

emitted light from the whole specimen

excitation light

3D specimen

Confocal microscopy excites the whole

specimen but collects emitted light

primarily from the focal point

3D specimen

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A typical research confocal microscope

Confocal

Conventional

The result is an image with reduced haze,

improved contrast and better resolution

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3D images can also be generated

Dim specimens are harder to image by confocal

because the pinhole rejects a lot of light

In practice, the pinhole is often made larger to

generate “partially confocal” images

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Partially confocal images are a compromise

conventional fully confocal

(small pinhole)

partially confocal

(medium pinhole)

partially confocal

(large pinhole)

Conclusion

Confocal microscopy can generate high contrast

3D images of specimens.

It has been the predominant instrument for high

resolution light microscopy in the past 10 years.

Dimmer specimens are more challenging.

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Detector

Computer

Microscope

Microscopist

Easy, computer-controlled time-lapse imaging

Cells are not only 3D, they are also alive.

How do we image changes over time?

Shutter

But the cells need to be happy too.

Sophistication of the chamber depends on the

sensitivity of the cells and the duration of the imaging

experiment. pH and temperature are key variables.

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Sometimes the cells are very happy

(But our budget is not)

Even with perfect incubation conditions,

repeated light exposure causes problems

heating (?)

photobleaching

dimmer signal

free radicals

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Photobleaching is always a problem

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4 5

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Bleaching rate depends on the dye

Ale

xa

Flu

or,

Cy3

Dyes have been optimized for bleaching and for the

laser lines available on typical confocal microscopes

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How are living cells labeled with dyes?

cell permeable dyes

microinjection of dyes

endogenous dyes

Cell permeant dyes

Fura2

Cleaved by non-specific esterases in the cell to

become impermeant, and locked in the cell.

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Fertilization-induced calcium wave in a starfish

oocyte. Confocal images every 5 sec

Calcium concentration can then be

measured in space and time

Microinjection

microinjection

needle

patch-clamped neural cell

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Microinjection is difficult and

time-consuming

Only a limited number of cells can

be injected per experiment (~100)

Endogenous probes are now the most

convenient and most widely used

Crystal structure of GFP – green fluorescent protein

-barrel

chromophore

27 kD

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Proteins of interest can be tagged with

GFP and observed in live cells

Gene for Your Protein GFP

Transform, transfect cells

Translocation of the GFP-tagged glucocorticoid

receptor from cytoplasm to nucleus

Surprising features often discovered by time-lapse

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Double or triple labeling of live cells is

feasible with GFP variants

A spectrum of markers: blue, cyan, green, yellow, red

Time-lapse imaging: a case study

from Molecular Biology of the Cell (1998) Bray et al.

How is chromatin packaged at the Mbp level

in the interphase nucleus?

And how does this packing change when a

region becomes activated?

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Cells contain ~200 tandemly repeated copies of

{MMTV - ras - BPV} and are stably transfected with

GFP-GR under tetracycline regulation.

Live-cell imaging done in a specially

developed cell line

The tandem array is visualized as a bright structure

above the background of GFP-GR in the nucleus

GFP-GR RNA FISH OVERLAY

These bright structures are the tandem array

because they produce the predicted transcript.

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How does this structure change over time as

transcription is induced?

Method: Time-Lapse Imaging

Problems

Very rapid bleaching

Cells become sick after only 10-20 images

Cells drift out of focus

Changes occur on slow time scale (~4 h) but need ~100 movies

Solutions

2D time lapse, 1 image every 15 min

Non-confocal images

Microscopist manually corrects focus during experiment

~5 data sets collected in parallel

Tools: Chambered cover slip, stage heater, conventional

fluorescence microscope with xy controlled stage

The microscope set up

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Results

Unfolding and refolding suggests fiber

packing at different densities

Conclusion

GFP and its variants have made it possible to

easily tag proteins with fluorescent markers, and

then observe the behavior of specific cellular

structures over time.