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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Interference
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principle that lenses use to focus light
Used in contrasting techniques
light waves can subtract and add
Polarization- allowing only light of a particular
vibrational plane
Refraction
Diffraction
Interference
Constructive
Destructive
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
Resolution vs.
Magnification
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(b)
(Fig 17.3)
(c)
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Ocular lens
Objective lens
Specimen
Condenser lens
Light source
Limit of Resolution
Resolution = 0.61l/nsinq = 0.61l/NA
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The cone of light collected by the lens
determines the resolution (nsinq)
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
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Plane of focus
of image
Plane of focus
of light source
 q
Plane of specimen
Lamp
(
) Light rays that form the image
(
) Background light of the field
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
l=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
ribosome- 20-30nm
protein2-100nm
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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Brightfield
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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
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
Hemotoxylin/Eosin stained tissue (Lodish 5-7)
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
Light source
l
4
l
2
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
Brightfield
DIC
Phase Contrast
Darkfield
C. elegans (worm)- Transparent tissue allows cells
throughout to be imaged by DIC (Figure 5-16, Lodish)
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
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http://www.olympusmicro.com/primer
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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 themdestroys 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
NA4
brightness=
mag2
a lens of equal magnification
but 2x NA will be 4x as
bright
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
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Monitor interactions between two proteins. Left: CFP-NIPP1, center:
YFP-PP1, right: FRET. Top: Both YFP-PP1g are expressed. NIPP1 binds
and retargets PP1 to nuclear speckles outside of nucleolus. Bottom:
Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget
speckles from nucleolus. After bleed-through correction, minimal FRET
can be observed (right). Images acquired during 2002 FISH Course
CSHL Labs (Universal Imaging Website).
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
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http://www.microscopyu.com
Specimen illumination
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Results
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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
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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
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 nonfluorescent sample, interaction of the
light with certain structures will cause it
to be converted to 480nm light
Works mostly with polarizable materials
like filaments
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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
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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” (antirabbit or mouse) to localize the primary
antibody
Immunofluorescence Visualization of Cell Structures
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.
Liver specific promoter
DNA
GFP gene
Protein on Liver
GFP
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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!
Liver protein gene
GFP gene
Liver protein
GFP protein
Allows you to do live cell dynamic localization of specific proteins
DNA
Protein
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Amoeba cells expressing GFP-Coronin fusion protein (green)
phagocytosing (engulfing and eating) yeast (red)
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Indirect visualization of actin filamentsGFP fusion with an actin binding protein
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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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l = 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 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!