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 Interference Limitations light waves diffract at edges-smearing causes limits resolution = minimum separation of two objects so that they can both be seen Limit of Resolution 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 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 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 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) Phase 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 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) Evanescent Wave Specimens 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 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 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 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 Specimen illumination Results 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 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 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 Anti-tubulin Immunofluorescent localization of microtubules
Green Fluorescent Protein (GFP)- An Ongoing Revolution in Cell Biology 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 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 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 λ = 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!