Part 1: Introduction and Cell Components Flashcards
how does electron microscopy work
it uses a beam of accelerated electrons as a source of illumination
what type of electron microscope creates 3-D but may require special preparation of the sample
scanning electron microscope
what type of electron microscope offers the greatest magnification, high resolution in two dimensions, and uses a fluorescent screen
transmission electron microscope
what is the relationship between speed of an electron and wavelength of an electron
there is a direct correlation
what is the % of formaldehyde in aqeuous buffered formalin
37%
what is the pH of formalin
7.0 - neutral
what are four examples of formed elements seen in tissue after fixation
nucleoproteins
intracellular cytoskeletal proteins
extracellular proteins
membrane phospholipid-protein or carb complexes
what substance clears tissue of alcohol before it is imbedded into paraffin
xylene
how thick are usual histologic sections
typically 4 microns
what is the common mounting media used to coverslip tissue
paramount
what is the first stain used in the staining process
hematoxylin
what counter stain is used in the staining process
eosin
what salt additive is used as a mordant to make hematoxylin act as a basic dye
aluminum
eosin attaches to what charged components in a cell
catatonic, + charged
what is a heavy metal that can be used to bind with lipids in the tissue commonly used in EM
osmium tetroxide
what is a good preservative to use when submitted tissue for EM
glutaraldehyde
what is a common transport media for live cells, especially lymph nodes
RPMI
what temperature is used for a cryostat
-15 to -20 degrees celsius
what methods of snap freezing causes the least amount of damage
isopentane
what is the most common method of snap freezing
liquid nitrogen (-196 degrees celsius)
what staining technique may be used to identify lipids during a frozen section
thionine
what stain is used for a parathyroid frozen
oil red-o
what is the name of the part of the antibody that binds to the antigen in IHC
epitope
what are the common reagents used for blocking in IHC
3% of both peroxidase and serum albumin
what are the three methods of labeling antibodies in IHC
flurochromes
enzymes
EM
cytokeratin identifies what
carcinoma and adenocarcinoma
vimentin identifies what
CT tumors and melanoma
LCA identifies what
leukemia and lymphoma
CD20 identifies what
B lymphocytes
CD3 identifies what
T lymphocytes
CD34 identifies what
angiomas, angiosarcoma, and fibroblastic tumors
SMA identifes what
myofibroblastic and myoepithelial tumors
desmin identifies what
muscle
S-100 identifies what
neutral cells, cartilage, granular cell tumors, and melanomas
HMB-45 identifes what
some melanomas
what is the name for the study of tissue structure
microanatomy
what are the 5 ways electrons react to an object in electron microscopy
unscattered (go right through)
secondary electrons
auger electrons
form an x-ray
back scattered
what are some disadvantages of electron microscopy
expensive
large machine
sensitive to things like vibrations
specimen must be in a vacuum chamber
needs a constant voltage supply
needs circulation of cool water
needs personnel training
what are some advantages of anatomic force microscope (a type better than electron microscopy)
sample preperation is easier
works in a vacuum, air, and in liquids
living systems can be studied, no fixation needed
higher resolution then TEM
what are some disadvantages of anatomic force microscope
limited vertical range
limited magnification range
tip or sample can be damaged
why is super resolution microscopy so affective
allows for visualization of subcellular organization with lots of details
what are the three main steps associated with routine tissue preparation
formalin fixation
paraffin embedding and cutting
staining
what properties does formalin fixation provide
terminate cell metabolism
prevent enzymatic degradation of cells by autolysis
kills pathogens
hardens tissue
maintains general structure of cell
what process must occur to tissue on a slide before it can be stained
dissolve paraffin with xylene
rehydrate tissue using decreasing alcohols
how is water removed after washing the formalin from the tissue
using a series of dehydrating alcohol
why must the tissue go through a series of alcohols before staining with eosin
eosin is more soluble in alcohol than water
which stain will show nuclear DNA and cytoplasmic RNA more
hematoxylin
hematoxylin attaches to what charged components in a cell
anionic, - charged
what is an example of a negatively charged component (basophilic) in a cell
nucleus
what is an example of positively charged component (acidophilic) in a cell
proteins in the cytoplasm
what does basophilic mean in tissue staining
tissue readily stains with basic dyes such as hematoxylin
what does acidophilic mean in tissue staining
tissue readily stains with acid dyes such as eosin
what is a good preservative when submitting skin for immunofluorescence
Zeus (Michele’s media)
why would you do frozen section on tissue
no preoperative diagnosis
unexpected intraoperative finding
evaluation of surgical margins
what media is used in performing a frozen section
OCT (optimum cutting temperature)
what are the three steps to prepare a frozen section
freeze tissue in OCT
cut tissue in cryostat
stain tissue on a slide and cover slip
immunohistochemistry targets antigens within the tissue by applying what substance
antibodies tagged with visual markers
what are some uses of immunohistochemistry
identify replicating cells, signaling cells, apoptotic cells, activation states, and different types of cells within the same tissue
what are the benefits of formalin fixation in IHC
antibodies preform optimally
prevents degradation of tissue
preserves position of antigen
what is the advantage of freezing tissue in liquid nitrogen or isopentane for IHC
the antigen is preserved
what is the disadvantage of freezing tissue in liquid nitrogen or isopentane for IHC
freezing artifact alters cellular morphology
what are two methods of antigen retrieval for IHC
HIER (heat inducted epitope retrieval)
enzyme digestion
what is the main purpose of the antigen retrieval process in IHC
to increase the accessibility of the antibody to the antigen
what is the purpose of permeabilization in IHC
to improve antibody penetration to access nuclear and cytoplasmic antigens
what is the purpose of blocking in IHC
to eliminate background staining
what are some disadvantages of using direct IHC techniques
less signal amplification so may not get a signal with little antigen
conjugation process may interfere with the antibody to react with target antigen
what is the main advantage of indirect IHC
sensitivity of antibody reacting to the target antigen is increased because multiple secondary antibodies can bind to a single primary antibody amplifying the signal
what are the main differences between monoclonal and polyclonal antibodies used in IHC testing
monoclonal are consistent from batch to batch but may have false negative results
polyclonal tend to have non-specific reactivity and vary from batch to batch
how are the results of IHC testing validated
using known positive and negative controls
what are some advantages of enzymatic methods of IHC
they can be viewed through a standard light microscope
enzyme table specimens have an unlimited shelf life
what are some disadvantages of enzymatic methods as compared to fluorescent methods in IHC
fluorescence has better resolution
enzyme intubation process causes testing to take longer
enzyme amplification interferes with signal quantization
substrates used to activate enzymes can be toxic or carcinogenic
positive controls are useful in IHC in detecting false negatives caused by what
poor fixation of tissue
problem with tissue processing
what are negative controls in IHC useful in detecting
endogenous biotin
peroxidase activity
what information does IHC provide a Pathologist
rendering a diagnosis when morphology alone isn’t enough
differentiation of a tumor
often used as prognosis and predictive markers in therapeutic treatment
what microbiology technique is preformed on cultured cells or embryos to localize specific nuclear sequences as small as 10-20 copies of mRNA or DNA
in situ hybridization
what microbiology technique is used to detect minute quantities of rare or single copy number nucleic acids sequences in frozen or embedded cells or tissue sections for the localization of those sequences within the cells
PCR
what is a single-stranded nucleic acid employed in the hybridization, requiring a color molecule to attach to it
a probe
what is used in the amplification of a sample in a hybridization technique that is significantly shorter than a probe
a primer
what type of test is used to determine trace amount of DNA in a sample
PCR
what type of test is appropriate for determining if there are antibodies present in the blood as a result of exposure
serology testing
