Microscopy & Methodology Flashcards
Resolution (Resolving distance. RD)
Smallest distance between two particles where they are seen as separate objects.
Magnification (enlargement)
Image size divided by actual size. For example, if the image size of an organelle is 1mm and its actual size is 1µm, then, the magnification is 1000X (1mm/1µm = 1000)
Human eye (R)
0.5mm
Light Microscope
Can see up-to 0.5micrometers. Maximum magnification=1000x
Use light for tissue illumination
Types:
Bright field, confocal microscope and flourescence microscope, phase contrast
Bright Field
Used for routine histopathological examination.
Fixation, embedding and staining (H&E) of tissues is usually required.
Phase Contrast
Mostly used for examining living, unstained cells
(sperm motility, dividing cells in tissue culture, etc.)
Confocal Microscope and Fluorescence Microscopes
Mainly used in research labs
Electron Microscope
Can see up to 0.5nm; Maximum magnification=400,000x
Use electrons released from metal
Types:
Scanning, Transmission
Transmission
Used for in-depth examination of cell components.
Fixation, embedding and staining (uranyl acetate and lead citrate) of tissues is required.
Scanning
Used for examining three-dimensional views of surfaces of cells, tissues, and organs.
Tissue Preparation for Microscopic Examination
- Dissect/Collect
- Fixation
- Dehydration
- Clearing
- Embedding/ Infiltration
- Sectioning
- Staining
- Resolution
Collection/ Dissection
Dissect a small piece of tissue: (5mm thick for paraffin and 1mm thick for epoxy sections (light and electron).
LM:5mm thick, 1-3 cm long
EM: 1mm Cube
Fixation
Preservation: Formaldehyde 4% for routine light microscopy, paraffin sections. Glutaraldehyde 2% followed by osmium tetroxide 1% for epoxy sections. Fixatives harden tissues and prevent autolysis.
Immersion fixation: For routine slide preparation by immersing tissues in a jar containing fixative that is roughly 20 times the volume of the tissue.
Intravascular perfusion: Fixative is injected through blood vessels, rapid and uniform fixation, but difficult to undertake, compared to immersion fixation.
Note: Do not use water-based fixative for glycogen fixation because it is soluble in water. Instead, use 100%
LM:Formalin, Alcohol, Freezing
EM: Glutaraldehyde, OsO4
Dehydration
The objective is to remove water. It is done by immersing tissues in graded concentration of alcohol (50%, 60%, 70%, 80%, 90% and finally 100%). Acetone can also be used instead of alcohol. Lipids are also lost
Same for both LM and EM
Clearing
The objective is to make tissues transparent so that paraffin or epoxy can easily infiltrate into the tissue. Xylene, benzene, or chloroform are used as clearing agents.
LM:Xylene, Benzene, Chloroform
Same for LM and EM
Embedding/ Infiltration
The cleared tissues are embedded with paraffin for LM or epoxy for LM and transmission EM.
LM: Paraffin
EM: Epoxy
Sectioning
The objective is to cut sections thin enough so that light (for LM) or electrons (for EM) can penetrate the tissue to form an image.
5µm thick sections for paraffin LM; 1µm thick sections for epoxy light microscopy; 50-80nm thick sections for epoxy transmission EM.
LM: 5-7micrometer
EM: 20-80nm
Staining
Light microscopy of paraffin sections: Staining add colors for contrast; most used stain is hematoxylin and eosin (H& E).
Electron microscopy of epoxy sections: Staining increases electron density and contrast. Heavy metals (lead citrate and uranyl acetate are used to stain tissues and cell organelles.
Hematoxylin and Eosin stain (H& E)
“Hematoxylin
“Basic” dye and has a positive charge. Hence it stains those cell structures that are acidic and have a negative net charge. For example, nucleus and ribosomes. Structures stained with hematoxylin stain blue (basophilic, attraction for basic dye).
Hematoxylin and Eosin stain (H& E)
“Eosin”
“Acidic” dye and has a negative charge. Hence it stains those cell structures that are basic and have a net positive charge. For example, most cytoplasmic components (except ribosomes). Structures stained with eosin stain pink (eosinophilic, attraction for acidic dye).
Histochemistry/ Cytochemistry
Uses special stains to identify chemical components of cells or tissues
What stains Carbohydrates
Periodic Acid Schiff (PAS)
What stains Glycogen
PAS (Best carmine, glycogen only)
What stains Lipid
Oil red/ black sudan (frozen section)
Osmium (Paraffin section)
What stains Mitochondria
Succinate dehydrogenase enzyme
What stains Lysosomes
Acid phosphatase enzyme
What stains Golgi body
Silver nitrate
What stains Peroxisomes
Catalase enzyme
What stains Elastic Fibers
Wiegert’s elastic stain
What stains Reticular fibers
Silver Nitrate
Immunocytochemistry
It is a highly specific interaction between antigens and antibodies and is useful in identifying and localizing specific proteins (enzymes) and glycoproteins within cells. It is an important diagnostic tool for detecting the type of cancer and identifying cytoplasmic components at the molecular level.
Autoradiography
It is used to visualize synthesis of a particular component in a cell by using a radiolabel (isotope) tritium - H3 carbon- C14.
For example: 1) synthesis of proteins using radioactive amino acids,
2) synthesis of carbohydrates by using radioactive simple carbohydrates (glucose),
3) synthesis of fat by using radioactive cholesterol,
4) synthesis of DNA by using radioactive thymidine,
5) synthesis of RNA by using radioactive uracil.
