Microscopes and Microscopy
MCB 380
Good information sources:
Alberts-Molecular Biology of the Cell
http://micro.magnet.fsu.edu/primer/
http://www.microscopyu.com/
Approaches to Problems in Cell Biology
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Biochemistry-You can define a enzyme reaction and then try to figure what does it,
when, where and under what control
Genetics- You can make a mutation and then try to figure out what you mutated
Cell Biology- You can visualize a process and try to understand it- for instance cell
division was one of the earliest
Today- there are no distinctions. You cannot be just one thing, or be knowledgable
about one thing. You need to take integrated appoaches to problems using the
appropriate tools when needed. If you limit your approach, you limit your science
Properties of Light
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Reflection
Diffraction-scattering of light around edges of objects
Limits the resolution
Refraction- bending of light when changing medium (index of refraction)
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principle that lenses use to focus light
Used in contrasting techniques
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Interference
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Polarization- allowing only light of a particular vibrational plane
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light waves can subtract and add
Refraction
Interference
Limitations
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light waves diffract at edges-smearing causes limits
resolution = minimum separation of two objects so that they can
both be seen
Limit of Resolution
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The cone of light collected by the lens determines the resolution
(nsinθ) n=refractive index
Max NA is 1.4 (refractive index of oil)
Lenses range from 0.4-1.4 NA
Maximum magnification is about 1000x
Resolution of Microscopes
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Visible light is 400-700nm
Dry lens(0.5NA), green(530nm light)=0.65µm=650nm
for oil lens (1.4NA) UV light (300nm) = 0.13µm
for electron microscope
λ=0.005nm but NA 0.01 so =30-50nm
Sizes of Objects
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Eukaryotic cell- 20µm
Procaryotic cell-1-2µm
nucleus of cell-3-5µm
mitochondria/chloroplast- 1-2µm
ribosome20-30nm
protein- 2-100nm
Microscope Objectives
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complex combinations of lenses to achieve
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high magnification
low optical distortion
Low chromatic distortion
flat field
Contrast
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Cells are essentially water and so are transparent
In addition to resolution and brightness, you need to generate contrast to see
things
Two objects may be resolvable by the microscope, but if they don’t differ from
the background, you cannot see them
Contrast can be accomplished with staining or optical techniques
Microscope types
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Brightfield
Stereo
Phase contrast
Differential Interference Contrast
Fluorescence
Confocal
Electron
Transmission
Scanning
Atomic Force
Microscopes
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Stereo
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Different images are sent to the two eyes from different angles so that a stereo
effect is acheived. This gives depth to 3D objects
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Brightfield
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use a prism to send the light to both eyes
light passing through specimen is diffracted and absorbed to make image
Staining is often necessary because very low contrast
Phase Contrast
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A phase ring in condenser allows a cylinder of light through in phase. Light that is unaltered hits the
phase ring in the lens and is excluded. Light that is slightly altered by passing through different refractive
index is allowed through.
Light passing through cellular structures such as chromosomes or mitochondria is retarded because they
have a higher refractive index than the surrounding medium. Elements of lower refractive index advance
the wave. Much of the backround light is removed and light that constructively or destructively interfered
is let through with enhanced contrast
Visualizes differences in refractive index of different parts of a specimen relative to the unaltered light
Hemotoxylin Stain of Chromosomes (18.5)
Phase Contrast
Differential Interference Contrast or DIC or
Nomarski
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A prism is used to split light into two slightly diverging beams that then pass through
the specimen.
On recombining the two beams, if they pass through difference in refractive index
then one retarded or advanced relative to the other and so they can interfere.
By changing the prism you can change the beam separation which can alter the
contrast.
Also measures refractive index changes, but for narrowly separated regions of light
paths-ie it measures the gradient of RI across the specimen
Gives a shadowed 3D effect
Optically sections through a specimen
DIC beam Path
Interference Reflection Microscopy
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Looks at light reflected off the surface only.
By polarizing the light and then analyzing the resultant, can see differences in
height of reflecting surface.
If something is closely opposed to the glass surface, then it does not pass
through a new medium and when reflected back it is eliminated.
Altered light is left in and looks light while closely apposed is dark.
IRM Light Path
IRM Images
Total Internal Reflection Microscopy
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Light shined on a reflective surface at an appropriate angle will generate an
evanescent wave, a wave of energy propagating perpendicular to the surface
It only propagates about 100-200nm from the surface
Allows one to visualize events taking place near the membrane (exocytosis,
cytoskeleton)
Evanescent Wave
Specimens
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Live cells or tissuecan you see the structure in a live cell?
can you image the cell without damaging it with light?
Fixed-try to retain structure intact
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Glutaraldehyde- reacts with amines and cross links them-destroys 3D structure of many proteins
Formaldehyde-reacts with amines and cross links them
slower reaction, reversible, not as extensive
Methanol, acetone, ethanol, isopropanol- precipitate material- not as good for retaining structure
Rapid freeze (liquid helium)- then fix
Fluorescence Microscopy
Fluorescence Microscopy
Fluorescent dye- a molecule that absorbs light of
one wavelength and then re-emits it at a longer
wavelength
 Can be used alone or in combination with another
molecule to gain specificity (antibodies)
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Epifluorescence Microscope
Dead cells stained with a Fluorescent reagent (fluorescent phalloidin- a fungal toxin) to visualize
actin filaments
Endoplasmic Reticulum Stained with a synthetic dye that dissolves in ER
membranes
Brightness of an image
Discussion Problem
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Actin filaments are 8nm in diameter
We can see a single filament with phalloidin stain in fluorescence
microscope
The resolution limit of the microscope is 200nm
WHY CAN WE VISUALIZE THE FILAMENT??
Co-localization of Proteins
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FRET- Fluorescence Resonance Energy Transfer
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If the emission wevelength of one probe overlaps with the excitation wavelength of
another probe you can get resonance energy transfer
Non-radiative transfer- the energy is transferred directly from molecule to molecule
The two molecules need to be within 10 nm because the energy transfer falls off
with the 6th power of distance
You excite with the donor wavelength and measure emission at the recipient
wavelength
Co-localization of proteins
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FLIM-Fluorescence Lifetime Imaging
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When a probe is excited briefly, the rate of decay of fluorescence is
different for each probe-so if you have different probes in the cell you can
characterize them based upon lifetime
FRET-FLIM- measure the decay of the donor during FRET
Confocal Microscopy
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Fluorescence microscope
Uses “confocality” (a pinhole) to eliminate fluorescence from out of focus
planes
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Minimum Z resolution=0.3µm
Because you can optically section through a specimen, you can determine the
localization of probes in the Z dimension
You can also build 3D (4D) models of structures and cells from the data
Laser scanning confocal
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Uses a laser to get a high energy point source of light
The beam is scanned across the specimen point by point and the
fluorescence measured at each point
The result is displayed on a computer screen (quantitative data)
Laser scanning confocal Microscope
Specimen illumination
Results
Spinning Disk confocal Microscope
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Illuminates the whole field “simultaneously with a field of points
Captures images of the whole field at once with a camera
Much faster than LSCM
Can be viewed through eyepieces
Nipkow spinning disk
Two photon confocal microscopy
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A fluor like fluorescein normally absorbs a photon of about 480nm and emits one at about 530nm
If fluorescein absorbs two photons of 960nm near enough to each other in time so that the first does not
decay before the second is absorbed, it will fluoresce- 2 photon fluorescence
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Confocal microscope with a laser that emits picosecond pulses of light instead of a continuous beam is
used
Advantage
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960nm light penetrates farther into biological specimens
The density of light is very high at focal point, but low elsewhere, so damage to cell is less
You don’t need a second pinhole because excitation only happens at the focal point
Second harmonic Imaging
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Uses same instrument as 2-photon microscope
If you shine 960nm light on a non-fluorescent sample, interaction of
the light with certain structures will cause it to be converted to
480nm light
Works mostly with polarizable materials like filaments
How do we get fluorescent probes into cells
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Kill the cell and make the membrane permeable
Live cells
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Diffusion: some can cross membrane
Microinjection- stick and tiny needle through membrane
Trauma: rip transient holes in membrane by mechanical shear (scrape loading) or
electrical pulse (electroporation)
Lipid vesicles that can fuse with membrane
Transfect with fluorescent protein vector
Loading Cells (Alberts 4-59)
Types of Probes
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Some change intensity of fluorescence depending on pH or [Ca++]
Some bind specific structures
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ER
actin
Golgi
Plasma membrane
Mitochondria
Fluorescently labeled purified protein
Antibodies
Microinjected Fluorescent Tubulin in a live cell
Immunofluorescence localization of proteins in
dead/fixed cells
You can purify almost any protein from the cell (Biochemistry)
 Make an antibody to it by injecting it into a rabbit or mouse (primary antibody)
 Use the antibody to bind to the protein in the fixed cell
 Fixed cells can be made permeable so antibodies can get into interior
 Use a fluorescent “secondary antibody” (anti-rabbit or mouse) to localize the
primary antibody
Immunofluorescence Visualization of Cell Structures
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Anti-tubulin Immunofluorescent localization of microtubules
Green Fluorescent Protein (GFP)- An Ongoing
Revolution in Cell Biology
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Protein from fluorescent jellyfish
The protein is fluorescent
Now cloned, sequenced and X-ray structure known
If you express it in a cell, the cell is now fluorescent!
Use a liver promoter to drive gene expression, and you get a fluorescent liver! All cells in the liver make GFP which
fills the cytoplasm with fluorescence.
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Fuse the DNA sequence of a protein to the DNA sequence of GFP and the cell will express it and make a fusion
protein which has two domains. Wherever that protein is in the cell, you will see fluorescence!
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Allows you to do live cell dynamic localization of specific proteins
Amoeba cells expressing GFP-Coronin fusion protein (green) phagocytosing (engulfing
and eating) yeast (red)
Indirect visualization of actin filaments- GFP fusion with an
actin binding protein
Problem 2
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I purify a nuclear membrane protein which I find to be 165kD in size. I then
make an antibody to the protein. When I immunostain the cell, I get
fluorescence in the nuclear membrane and in the Golgi. When I run a Western
blot, I get a 165kD band and a 60kD band. Give two explanations to explain
the results and then describe what you would do to clarify the results.
How to get around the problem of resolution?
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Invent the Electron Microscope
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Uses electrons instead of light to form an image
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Wavelength of electron decreases as velocity increases so accelerated electrons have a very short wavelength
compared to visible light
You need to use magnets as lenses to focus the beam
View electrons striking fluorescent screen
TEM- Sees electrons that pass through the specimen. Electrons scatter when they strike the specimen so
as density of material increases, more electrons make it to the detector
SEM- Looks at the electrons reflected as a beam is passed over the specimen
Resolution
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λ = 0.004 nm
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If lenses were as good as optical ones, resolution would be 0.002 nm (100,000x better than light)
but NA of magnetic lenses is much worse so for biological specimens
resolution= 2 nm (100x better than light microscope)
Sample Preparation for EM
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Must be done in vacuum for electron gun to work
Can’t have water in vacuum!
Dry tissue does not have enough density to scatter electrons so you have to replace it with something
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dense.
Procedure
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Fix Tissue (glutaraldehyde or osmium)
Dehydrate and embed with plastic
Stain with Osmium, lead etc. or make metal replica
For TEM- Section (0.02-0.1µm thick)- so you only look at very thin section
For SEM- No sectioning- you only see the outer surface
What you see is the scattering of electrons by the metal. There is no biological material left!
Immuno-electron microscopy
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You can’t see antibodies in the EM
You can attach dense particles to antibodies to make them visible
Allows you to visualize the localization of specific proteins in the
EM
Very hard to do!