Microscope Lab Flashcards
Compound Light Microscope: Ocular Lens definition
The eye lens
Additional 10X mag
Compound Light Microscope: Objective Lens definition
Magnifies sample
4X, 10X, 20X, 40X
Compound Light Microscope: Objective Turret definition
Hold objectives
Compound Light Microscope: Stage definition
Location of slide placement
Compound Light Microscope: Slide holder definition
Holds slide snugly
Compound Light Microscope: Mechanical Stage Controls definition
X and Y movement
Compound Light Microscope: Course Focus
This can drive objective through slide
Compound Light Microscope: Fine Focus
Mostly use this one
Compound Light Microscope: Condenser
Focuses light on specimen
Compound Light Microscope: Iris diaphragm level
Controls light through condenser
Compound Light Microscope: Rheostat
Controls light intensity (on side of base)
Compound Microscope
- Has more than 2 sets of lens
- Considered binocular because it has 2 eyepieces rather than 1
- Allows you to alter the distance of the 2 eyepieces to accommodate your interpupillary distance to view single image
Resolution
Defined as the ability to distinguish 2 items that are closely spaced as separate entities
Resolving Power
- 200microm or 0.2mm
- Items closer than 200 microm from each other cannot be distinguished without optical assistance
Electron microscope resolving power
2-3 nm
Fixation
- Formaldehyde for LM
- Glutaraldehyde for TEM
- poor fixation = poor slide quality
- During fixation, cell water is replaced with a buffered (physiological pH) chemical that binds (fixes) proteins
- Most fixatives are aldehydes
- Critical parameter of time spent in fixture: 18-24 hrs
- Critical parameter (fixative volume to tissue volume ratio): 10X more fixative
Problems from Bad Fixation
- Excessive fixation time can cause the tissue to be brittle
- Too little time won’t let the fixative diffuse throughout the entire tissue sample
Processing
- Paraffin for LM
- Epoxy/resin for TEM
- Replacing the aqueous fixative solution within the cells with an embedding medium
- Tissue is dehydrated through an increasing graded series of alcohol (removes all the water from the cells and replaces it with ethanol)
- Add solvent that is miscible with the embedding medium (xylene for paraffin), (propylene oxide for eposy/resin)
- Sample is placed in pure solvent, then 50% solvent/50% EM & then 100% EM
Embedding
- Paraffin for LM
- Epoxy/resin for TEM
- Requires proper orientation of the sample within the embedding mold (cassette for LM; capsule for TEM) followed by polymerization/ curing of EM
- once sample is oriented properly within the EM, it is filled with the embedding media
- Paraffin embedding requires cooling @ room temp to solidify
- Epoxy/resin embedded requires polymerization with heat
- Final product = block
- **Once embedded, the sample is protected from degradation and blocks are stored @ room temp
Sectioning
- LM: 4-8 microm
- TEM: 60-90nm
- act of slicing the tissue block thinly and placing it on a glass slide (LM) or a copper grid (TEM)
- LM: microtome, stainless steel knife
- TEM: ultramicrotome, precision glass knives/diamond knives
Staining
- LM: hematoxylin & eosin
- TEM: uranyl acetate/lead citrate
- adding dyes/stains, antibodies, fluorescent probes to sections to make them viewable
- For LM, paraffin must be removed from the tissue (needs to happen or it will inhibit the cells fro taking up/reacting with stains & dyes)
- xylene is used to remove paraffin from the tissue section followed by rehydration through a decreasing graded series of alcohol from 100% alcohol/water
Intensity of Staining is due to:
- abundance of a macromolecule, structure, or component
- tissue section thickness
- staining method
- age of slide
Hematoxylin
- chemistry of dye: basic stain (acidophilic)
- chemistry of structures: acidic structures
- results on slides: nucleic acids (DNA & RNA)
- color: blue, purple
- **
- stains the nucleus (DNA), nucleolus, and cytoplasm in areas of abundant RNA (rER)
- Makes them visible through LM
- color of the nucleic acids stained with hematoxylin will range in the intensity from blue to purple to almost black
Pattern of Staining for Hematoxylin informs what:
1) cell metabolic activity
2) cell cycle & mitotic phase of cell
3) cell viability (apoptosis)
Eosin
- chemistry of dye: acidic stain (basophilic)
- chemistry of structures: basic structures
- results on slides: proteins
- color: pink, red
Pattern of Staining for Eosin informs what:
- Presence of connective tissue
- Muscle types
- Abundance of mitochondria
What are some other facts of H&E staining?
- With H&E staining, the hematoxylin will fade before the eosin so the old slide looks light pink in color
- An abundance of hematoxylin & eosin doesn’t stain tissue
Amphophilic
- having the same affinity for basic and acidic dyes
- example: neutrophil
- neutral in chemistry as the tissue and cell chemistry has acidic and basic structures
Trichrome
-stain collagen
-common recipes in this course: Masson’s trichrome & Mallory’s trichrome
-importance: provides contrasting color for collagen vs. other cells/tissues carrying various proteins
-stains collagen as blue/green
***
Masson’s trichrome staining:
-nuclei: black
-cytoplasm: pink, red, brown
-keratin, erythrocytes, myelin & muscle: red
-collagen: blue, green
Why is trichrome staining different from H&E?
- In H&E, all proteins stain at a different intensity of red as it’s hard to distinguish pink cardiac muscle (healthy) from pink collagen (scar tissue)
- With trichrome, college is specifically blue/green
Periodic Acid Schiff’s (PAS) Stain
- oxidizes hexose ring of monosaccharides to determine the location and abundance of carbohydrates
- macromolecules of note: glycogen, glycoprotein, proteoglycans
- Can be found in: (1) connective tissue, (2) glycocalyx of cells, (3) basal laminae of epithelial cells, (4) muscins of mucus-secreting cells and tissues
- demonstrates fungi and yeast cells
- reaction produces a HOT PINK/MAGENTA color
- hematoxylin is used as a counterstain to reveal the nuclei
Elastic stains
- stains elastin and elastic fibers
- Verhoff’s stains elastin as black
- Aldehyde-fuchsin stains elastin dark purple
- Orcein stains elastin bright pink/magenta
Silver stains
-reduce metal salts causing them to precipitate on certain structures and components within cells and tissues
-Example: silver salts
-Helps visualize the fine structure of the nervous system and connective tissues in delicate organs
-demonstrate fungi and spirochees
***
Golgi Stain
-originally used the gold salt, replaced with silver salt
-stained with silver salt is going to be black
**Was usually used in the nervous system pathways
Reticulin Stain
- reticular fibers are an extracellular matrix component formed by the Type 3 collagen
- Example of a silver stain used to demonstrate the complexity of the reticular network in various organs (esp. smooth muscle and lymphoid organs)
- this is a type of a silver staining method so the result would be black precipitate on the reticular fluids
Total Magnification
Objective magnification X ocular lens magnification (always 10X)
Field of View
-circle you look through the eyepiece of your LM
-field numbers (located on side of ocular lens) = 16-22
-always in mm
*******
diameter of FOV = field number/objective we have in place
Lumen
middle/opening of the hollow, tubular organ (blood vessel, intestines) that substances pass through
Artifacts
- aberrations on slide
- some of these could be due to processing (different rates of shrinkage can cause some tissues to separate from another to create an artificial space
- sectioning errors: knife marks
- section placements: wrinkles are common, especially in large slides
- staining: dye can fall out of solution and collect on tissue
- cover slipping: air bubbles from dirty coverslip
Interphase nucleus
- pale white/gray areas contain euchromatin (open DNA that can be actively used in the cell)
- Under H&E, these same areas would stain very little and appear clear to very pale blue
- the dark black areas are heterochromatin (DNA not actively transcribed)
- Under H&E, these same areas would stain as dark blue and purple
- Some of the heterochromatin represents the redundant DNA abundant in eukaryotes
- The pattern of euchromatin and heterochromatin, its ratio can change for a cell type based on its metabolic activity
Endothelial cell nucleus
- marginated heterochromatin pattern
- the heterochromatin is packaged against the nuclear envelope
- the heterochromatin outlines the borders of the nucleus within the cytoplasm of this cell
- without the packaging, the nuclear edges within the cytoplasm of a cell will be vague due to the inability to resolve the nuclear envelope itself under LM
Metabolically active cell
- typical nucleus of a cell producing abundant protein
- Has euchromatic nucleus, prominent nucleolus, and some marginated heterochromatin
Nucleolus
- Sphere will stain blue and purple as it contains both RNA and DNA
- the ribosomal subunits exit the nucleus through nuclear pores within the nuclear envelope
Rough Endoplasmic Reticulum
- Ribosomes are the location where RNA is translated into protein
- All cells make proteins that either remain within the cytoplasm for the cell’s day to day operations or is secreted for other purposes
- the cytoplasmic proteins are translated on polyribosomes that are free within the cell’s cytoplasm
- protein is packaged and secreted is translated on rER
Mitochondria
- Under TEM, they look like striped tigers
- In H&E staining, an abundance of mitochondria will result in a deeply red cytoplasm
