Basic Histology
8.25.04
Introduction to Microscopy
Outline:
I. Light Microscopy
II. Electron Microscopy
III. Cell Fractionation
IV. Cell Culture
V. Tissue Divisions
Introduction:
Histology: The study of structural and functional relationships within and between tissues and their cellular constituents
-requires microscopic methods of study
I. Light microscopy:
A. Standard Light Microscopy:
a) microscope design:
i) light source
ii) condenser
iii) object (specimen)
iv) objective lens
v) ocular lens (eye piece lens)
b) resolution
i) determined by the wave nature of light
ii) determined by the numerical aperture of the condenser and objective lens
iii) about 0.2 micrometers when everything is correctly adjusted
c) sample preparation:
i) fix: stabilizes cellular constituents (proteins and polymeric nucleic acids)
- chemically fix:
- precipitate:
ii) dehydrate/embed: infiltrate tissue with solid material to allow for cutting
-dehydrate in ethanol/xylene (removes lipids)
-infiltrate with paraffin or plastic
-can freeze to solidify tissue for sectioning (cryosectioning)
iii) section: cut tissue very thin
-typically 1 - 10 micrometers thick
-paraffin/plastic embedded tissue sectioned with micro tome
-frozen samples sectioned with cryostat (micro tome within a freezer)
iv) issues:
-paraffin/plastic sections:
-loose most enzyme activity and most protein function
-distortion of tissue
-loss of some constituents
-frozen sections:
-enzyme activity is usually maintained
-tissue can degrade upon thawing
-distortion of tissue
-ice crystal damage to specimen
d) means to impart contrast to specimen through illumination method and/or staining
i) non-stained specimens: contrast is low unless special optics are used
-bright field illumination: most common but contrast is very low unless specimen absorbs/diffracts a lot of light
-phase contrast: no stain, special optics that use differences in refractive index between specimen and surround to enhance contrast
-interference contrast: no stain, similar to phase contrast but 3-D appearance
-dark field illumination: specimen may or may not be stained, uses light scattering to impart contrast to the specimen
-fluorescence illumination: natural fluorescence materials in specimen or specimen stained with fluorescent dyes; illuminate sample at one wavelength and observe light emitted at a higher wavelength by the fluorescent material.
ii) stain specimen with dyes (classical histology): paraffin is removed from section then section is re- hydrated and stained
-some classical histological stains:
-hematoxylin: basic dye (+ charge), binds anionic (- charge) moieties
-eosin: acid dye (- charge), binds cationic (+ charge) moieties
-periodic acid-Schiff (PAS): chemical reaction that imparts stain to carbohydrates
-Masson's trichrome: cocktail of dyes
-Alcian blue:
-Reticulin stain: silver staining method
-Nissl stain (methylene blue):
-Sudan black/Oil Red O/Osmium tetroxide:
e) other methods methods to impart contrast to specimen:
i) enzyme histochemistry
-requires enzyme activity within specimen
-enzymatic reaction coupled to reaction that generates insoluble product (ppt)
-used clinically for various genetic disorders, used biologically to determine muscle fiber type (both metabolic and myosin isoform)
ii) immunocytochemistry: use specific antibody to localize specific antigen in specimen
-detect antibody via:
-coupling to enzyme that generates insoluble product
-coupling to a fluorescent molecule
-coupling to a gold or other electron dense particle
-coupling can either be direct (to the Ab) or indirect (to and Ab against the 1st Ab)
iii) autoradiography:
-radioactive substance incorporated into sample
-sample covered with photographic emulsion
-isotope decay results in silver deposits very near the site of the isotope
-used primarily with in situ hybridization to localize specific mRNA
B. Confocal scanning light microscopy (a form of fluorescence light microscopy):
a) microscope design:
- similar to light microscope in terms of basic design
- fluorescence excitation is via a laser that is scanned across the specimen
-very small region excited minimizing light scattering artifacts
-emitted light is passed through a pinhole (small aperture) prior to detector further reducing the signal from light scattering
-emitted light is detected via a photo multiplier tube to amplify signal and this is converted into an intensity signal for a specific x y coordinate
-rastering across the specimen gives a series of intensities at each x,y coordinate within the field and this is converted to an image on a monitor
-MAJOR advantage of being able to collect images within an optical plane (slice of the specimen) with minimal noise from signal above and below the optical plane ("higher resolution image")
-can obtain images at various regions through the thickness of the specimen and reconstruct a Z-axis image of the specimen
C. Digital Light Microscopy and Image Analysis/Manipulation
a) used for both normal and scanning (confocal) light microscopy
b) image is collected by camera attached to microscope or via detector (PMT of confocal)
c) output is a data set of the intensity at each x,y position (pixel) in the field (8, 10, 12, or 16bit)
d) this digital data can then be analyzed to measure how much and where the signal is coming from within the specimen
e) image can be manipulated digitally to enhance contrast
f) developed software can "de convolute" and standard microscope image into a confocal-like image
II. Electron microscopy:
A. Transmission electron microscope:
a) microscope design: similar to light microscope but use electrons instead of photons, image reconstructed from differences in electron scattering and absorption by specimen.
b) resolution: 0.1 to 1 nanometer
c) sample preparation:
-fixation: more robust methods, glutaraldehyde and osmium tetroxide are the fixatives
-dehydration/embedding
-dehydrate in graded ethanol/solvents
-embed in plastic (very hard)
-section
-very thin, 10 - 100 nm
-section placed on carbon coated copper grid
-stain: impart contrast (electron dense stain)
-sample can be prepared using freezing methods
-requires very cold temperature and thin section
-frozen sections dehydrated then coated with metal
-metal imparts contrast and mimics structure
-may be more like true biological structure
d) methods to impart contrast:
-positive staining: use heavy metals (electron dense salts of uranium and lead), most common method for tissue sections
-negative staining; use heavy metals (salts of uranium, tungsten, and molybdenum) to stain suspensions of isolated proteins or macromolecules, specimen is electron lucent in matrix of electron dense metal
-metal shadow casting (rotary shadowing, metal replicas): coat freeze dried sample with metal (gold, palladium, platinum, tantalum, tungsten), remove sample, observe metal "cast" of sample, gives topography of the sample
-immuno-electron microscopy (immunostain to give specific signal in specimen)
e) specialized methods:
-immunoelectron microcopy: similar to light microscopy but contrast via electron scattering differences
-immunostain using gold-labeled Ab's (gold is good electron scatter)
-immunostain sample then process for EM with positive or negative staining, Ab adds extra density (+ ve stain) or opacity (- ve stain) to specimen
-immunochemistry: immunostaining coupled to a reaction that generates and electron dense product
-autoradiography: similar to light microscopy methods with silver being an electron scatterer, can follow temporal pathway of molecule through a cell
B. Scanning electron microscopy:
a) microscope design:
-image constructed from reflected and emitted electrons
-image has 3 dimensional appearance as if viewed macroscopically
b) sample preparation:
-samples fixed but usually not sectioned
-samples coated with metal that reflects electrons
III. Cell fractionation
(reductionist approach)
a) tissues ground up and subjected to differential centrifugation or density gradient centrifugation
b) can isolate different cellular constituents for study at the biochemical or structural level
IV. Cell culture:
a) important tool for studying pure cell populations
b) allows for microscopic observation of living cells
c) allows for following differentiation of cells during development
d) study disease processes/cells
e) cells typically de-differentiate when in culture
V. Tissue Divisions:
a) Epithelia; covers ya
b) Nerve communicates with ya
c) Connective: keeps ya together
d) Muscle; moves ya and things within ya
e) all organs are made of these 4 tissues, one tissue is usually the dominant (by volume or mass) within a particular organ
