Intro to Histology and Cytology 8-26 Flashcards
Anatomy roots
apart, to cut
Macroscopic
Gross anatomy
Microscopic anatomy
Cytology, Histology, Organology Now histology is all of these
Cytology
Cells too small for naked eye to study
Histology
Tissues (epithelial, connective, etc.)
Organology
Microscopic nature of organs.
Function always reflects Structure
Physiology always reflects Anatomy
Organ system level
Cannot survive alone
Light Microscopy
Extremely thin slices. Specimens examined via transillumination (light going through them).
Electron Microscopy
Greater resolution, higher magnification - two types (TEM, SEM). Electrons and tissue, uses electromagnetic field as lens.
TEM
Transmission Electron Microscopy - in a vacuum, uses a beam of electrons that passes through the specimen
SEM
Scanning electron microscopy - beam of electrons scans the surface of the protein.
Atomic Force Microscopy
Gets down to molecular and atomic resolution. DNA can be viewed. A probe 1 atom thick passes over surface. When it is deflected it deflects a laser and produces an indirect scan. No vacuum, can use live specimen.
Resolving power
How far two objects must be separated from one another so that they can be distinguished.
Human Eye reso
0.2 mm - set by space in rods and cones in retina.
LM reso
0.2 microm - can’t see ribosomes, membranes, actin.
SEM reso
2.5 nm - in vacuum
TEM
0.05 nm (theoretical) - 1.0 nm practically on tissue
Atomic Force Microscopy
50 pm
Resolution is dependent on…
Optical system, wavelength of light source, specimen thickness, quality of fixation, staining intensity
Steps for LM
- Acquisition of cells, 2. fixation, 3. processing, 4. Embedding. Some distortion occurs along this process.5. Sectioning (cut b/w 1-15 microm), 6. Staining (reverse of what they did before getting rid of wax).
Processing
Preparing tissue to be embedded. 3 steps - dehydration, clearing, infiltration. Idea is to encase in parafin wax to be able to cut a thin slice. Need hard tissue.
Dehydration
Using a graded series of alcohol. - wax doesn’t mix with H20 so it needs to be removed. 3+ steps.
Clearing
Using a miscible substance - xylene in this instance. This helps parafin wax penetrate cells.
Xylene
Organic substance that is miscible with parafin. Causes tissue to almost be clear.
Side effects of LM fixing
Stops metabolism, distorts tissue, avoids autolysis, kills bacterial viruses, hardens tissue. Takes about 12 hours depending on thickness of tissue.
Gluteraldehyde, Formaldehyde
Fixation - good to preserve protein crosslinks (stops enzymes) and kill all.
Infiltration
58-60 degrees, allows wax to enter cells.
Staining
Reverse of what they did before to get rid of wax.
Problems with LM fixing
Long time - 2.5 days, solvent dissolves liquid, shrinkage of tissue.
Cryostat
Freezing in liquid nitrogen. Fast - used post surgery. Tissue placed in embedding medium and frozen, cut and stained. Enzymes not destroyed, stain can detect them.
Double fixation
First uses gluteraldehyde, then osmium tetroxide.