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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 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 Reflection Diffraction-scattering of light around edges of objects Limits the resolution Refraction- bending of light when changing medium
(index of refraction) principle that lenses use to focus light Used in contrasting techniques
Interference light waves can subtract and add
Polarization- allowing only light of a particular vibrational plane
Refraction
Diffraction
Constructive
Destructive
Interference
Limitations light waves diffract at edges-smearing
causes limits resolution = minimum separation of two
objects so that they can both be seen
Resolution vs. Magnification
(a) (c)(b)
(Fig 17.3)
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Ocular lens
Specimen
Objective lens
Light source
Condenser lens
Limit of Resolution
The cone of light collected by the lens determines the resolution (nsin) 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 = 0.61/nsin = 0.61/NA
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2
Lamp
Plane of specimen
( ) Light rays that form the image
( ) Background light of the field
Plane of focus
of light source
Plane of focus
of image
Resolution of Microscopes 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 =0.005nm but NA 0.01 so =30-50nm
Sizes of Objects Eukaryotic cell- 20µm Procaryotic cell-1-2µm nucleus of cell-3-5µm mitochondria/chloroplast- 1-2µm ribosome-20-30nm protein- 2-100nm
Microscope Objectives complex combinations of lenses to
achieve high magnification low optical distortion Low chromatic distortion flat field
Contrast 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
Brightfield Stereo Phase contrast Differential Interference Contrast Fluorescence Confocal Electron Transmission Scanning Atomic Force
Microscopes Stereo
Different images are sent to the two eyes from different angles so that a stereo effect is acheived. This gives depth to 3D objects
Brightfield 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
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)
2
Light source
λ
4Phase Contrast
Differential Interference Contrast or DIC or Nomarski
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 Phase Contrast
DIC Darkfield
C. elegans (worm)- Transparent tissue allows cells throughout to be imaged by DIC (Figure 5-16, Lodish)
Interference Reflection Microscopy
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
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)
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Evanescent Wave
http://www.olympusmicro.com/primer
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Specimens Live cells or tissue- can you see the structure in a live cell? can you image the cell without damaging it with light? Fixed-try to retain structure intact
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)
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
a lens of equal magnification but 2x NA will be 4x as
bright
NA4
mag2brightness=
Discussion Problem 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 FRET- Fluorescence Resonance Energy
Transfer 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).
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Co-localization of proteins FLIM-Fluorescence Lifetime Imaging
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 Fluorescence microscope Uses “confocality” (a pinhole) to eliminate
fluorescence from out of focus planes 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 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 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 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 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 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
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How do we get fluorescent probes into cells
Kill the cell and make the membrane permeable Live cells
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 Some change intensity of fluorescence depending on
pH or [Ca++] Some bind specific structures
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
Break Cells
Purify nuclei
Purify nuclear membrane proteins
Inject into rabbit
Prepare serum from rabbit with antibodies to protein
Link fluorescein to Antibody or
buy fluorescein linked anti-antibody (goat anti rabbit)
Immunofluorescence Microscopy
Anti-tubulin Immunofluorescent localization of microtubules
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.
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!
Allows you to do live cell dynamic localization of specific proteins
GFP protein
Green Fluorescent Protein (GFP)- An Ongoing Revolution in Cell Biology
GFP gene DNA
GFP Protein on Liver
DNA
Liver protein Protein
GFP gene
Liver specific promoter
Liver protein gene
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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 filaments- GFP fusion with an actin binding protein
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Problem 2 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?
Invent the Electron Microscope Uses electrons instead of light to form an image
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 = 0.004 nm
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
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
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 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!