Lecture 2: Microscopes, Prep, & Staining Flashcards
major components of microscopes
light source
condenser
stage
objective and ocular lens
pros of compound microscopes
magnify up to 1000x
allows you to see structural detail in high resolution
resolution is defining factor of high magnifications
cons of compound microscopes
specimens must be very thin
if not stained, everything is the same color
converts phase shifts in light passing through a transparent specimen into brightness
phase contrast microscope
advantages of a phase contrast microscope
allows you to see features of a transparent specimen as if it weren’t transparent
can examine unstained specimens
useful for observing living specimens
detects molecules that emit light, in wavelengths that lie within the visible range when exposed to a UV light source
fluorescence microscope
a naturally occurring fluorescent molecule
vitamin A
through the addition of a pinhole, optical resolution and contrast are increased
confocal scanning microscope
how does a pinhole increase resolution and contrast?
by eliminating out of focus light
with a confocal scanning microscope we can use a computer to ______ ?
reconstruct 3D images
advantages of a confocal scanning microscope
computer can eliminate out of focus material
can create images of very thin specimens (1 micrometer)
computer creates 3D image
what does TEM stand for?
transmission electron microscope
how does a TEM work?
uses a beam of e- instead of light waves
e- are driven by electromagnets from source to specimen
and then image is projected onto a viewing screen and photo film
what is the major disadvantages to TEM?
very large
very expensive
list the basic steps in tissue fixing and embedding
fixing
dehydration
alcohol removal
embedding
describe ‘fixing’
prevents further deterioration and helps to harden the tissue prior to embedding but can radically distort the specimen.
what is the most common fixative?
formalin
acid fixatives are used to see…..?
fix chromatin, nucleoli, spindle fibers
basic fixatives are used to see…..?
mitochondria
chromatin are destroyed
Carnoy’s Fluid
acid fixative
for preserving glycogen
Zenker’s Fluid
for sharp histological details
but if not washed out black crystals will form
acid fixative
Bouin’s Fluid
cytological details
uses picric acid which is extremely volatile
acid fixative
Zirkle-Erliki
basic fixative
long fixing time ~2days
glutaraldehyde
fixative for TEM
preserves proteins
osmium tetraoxide
fixative for TEM
reacts with lipids
gives density to structures in specimen
describe the ‘dehydration’ step
all water is removed through putting samples in ascending alcohol strength baths
what is the result of using ethanol baths with fat cells
dissolves neutral fats, so fat cells appear empty
why must we utilize dehydration when preparing slides?
all water must be removed because it will be embedded in hydrophobic materials
what is the next step after dehydration?
hydration
removal of alcohol used to dehydrate specimen
how do we removal ethanol from specimen?
replace alcohol with xylene or cedar oil
how does xylene effect specimen prep?
xylene removes alcohol but…
renders specimen transparent
why must we remove the alcohol from a specimen?
embedding materials, such a paraffin wax, will not mix with alcohol
what is the purpose of embedding a specimen?
turns it into a semi solid material that can easily be slicing for slide prep
describe the process of ‘embedding’
moved through paraffin baths – usually 3
this removes xylene
then placed in a mold with molten paraffin wax
which rapidly hardens in a cold water batha
what is used for embedding specimens for TEM?
infiltrate tissues with monomeric resin or epoxy resin
resin is polymerized
what is the purpose for fixing tissues?
because most things are transparent
what is the purpose for embedding tissue before sectioning?
biological material is squishy and mostly water
we must harden and embed material in a solid substance before we are able to precisely cut and section the specimen
advantages of formalin fixative
can be used alone or in combination
reacts with tissue proteins — stabilizing structures
cheap
easy to use
examples of what formalin can be used in combination with
alcohol — shrinks tissues
acetic acid —- softens and counteracts alcohols effects
disadvantages of formalin fixative
not good if you want to see fine cytological details
what is the purpose of the dehydration and hydration cycles used for tissue processing
dehydration removes all water because embedding materials are hydrophobic
hydration to keep specimen pliable and decrease deterioration —- organic material is not solid
contraption that uses a sharp blade over a stage that can be raised or lowered per desired slice width
microtome
what are the two types of microtomes discussed in class?
rotary
hand-held
how does sectioning differ for TEM specimens?
must use diamond knives
slices are 50 - 150nm thick
a copper mesh is used
what is a copper mesh used for in tissue sectioning?
in TEM, slices are so thin, they are too fragile to be handled. slice must be floated on a coated copper mesh
how does a copper mesh affect the viewing ability of a specimen?
copper mesh is left in forever
but we are viewing such a small area that the mesh holes allow e- to easily pass thru
why is it preferred that tissues be stained for observation?
because animal tissues are mostly colorless
staining allows us to see the details
what are the steps involved in staining a paraffin embedded specimen?
paraffin removal from section dehydration stain application dehydration alcohol removal add drop of cement cover slip
how is paraffin removed from section?
via xylene
first dehydration step in staining
must remove xylene that was used on paraffin removal
using series of alcohol to water — baths
then we can apply the stain
at what point in the steps of staining, is the actual stain applied?
after the first set of dehydration
what happens after the stain is applied to a specimen?
second round of dehydration
graded alcohol baths
after second dehydration phase of staining…..
alcohol is then removed with xylene
xylene is now admissible with mounting agent
what is the purpose of the cement drop ?
causes the outside of specimen to be solid and let the inside remain liquid like
what are the components of the most common staining technique for tissues in general?
HnE stain
hematoxylin and eosin
why is HE stain so common?
because displays structural features not chemical
staining properties of hematoxylin
behaves like a basic dye
helps stain stick to tissue
capable of staining nuclear material and cytoplasmic components
staining properties of eosin
an acidic dye
stains most cytoplasmic components and extracellular material
compares colors of HE stain
H — dark to light blue/purple
E — yellow to pinkish colors
react with anionic groups of tissue components
basic dyes
bind to tissue components by forming electrostatic links with cationic groups of tissue components
acidic dyes
any tissue component that reacts with a basic dye
basophilic
any tissue component that reacts with an acidic dye
acidophilic
examples of anionic groups of tissue components
phosphate, sulfate, carboxyl groups
examples of basic dyes
methyl green
methylene blue
pyronin G
toluidine blue
can pH affect staining color hues?
yes
examples of cationic groups of tissue components
amino groups of proteins
what is the purpose of using acidic dyes in sequence?
different acidic dyes can stains different cationic groups
specific sequences can be used to yield specific results pertaining to what you want to view
examples of acidic dyes
aniline blue
acid fuchsine
orange G
aniline blue
acid dye
stains collagen
acid fuchsine
acid dye
stains ordinary cytoplasm
orange G
acid dye
stains red blood cells
what does metachromasia refer to?
the phenomenon where dye changes color after reacting with a tissue component
examples of metachromasia
toluidine blue
stains cartilage ground substances or mast cell granules
what is the defining factor of TEM stains?
stains are ions of heavy metals that electron dense
examples of TEM stains
osmium tetroxide
uranyl nitrate
uranyl acetate = lead citrate
what does SEM stand for?
scanning electron microscope
SEM stains
platinum
gold
used to study the chemistry of cells and tissues
histochemistry
cannot use alcohol when viewing lipids
frozen specimens work great for adipose tissues
used to study the presence of specific constitutes by using monoclonal antibodies
immunocytochemistry
constitutes = antigens
proteins, glycoproteins and proteoglycans
antigens
monoclonal antibodies are derived….?
from activated B cell clones that have been exposed to the specific antigen
a single immune response to an antigen
monoclonal
B lymphocytes can mutate into _____, resulting in a _____.
tumor cells
myeloma
the 2 ways to label in immunocytochemistry
direct labeling
indirect labeling
types of direct labeling
flurorescent microscopy – fluorescent dye
light - visible substance
E-microscopy — gold or ferritin
describe how direct labeling works
conjugation with a type of direct labels, produces a visible marker
describe how indirect labeling works
the marker is attached to a second antibody, which is specific to the antibody used to locate the antigen of interest
this reagent rxn depends on the formation of aldehyde groups following the exposure to HCl or periodic acid
Schiff reagent rxn
mild hydrolysis with HCl exposes aldehyde groups on deoxyribose, which allows ________ . what is this rxn called?
Schiff reagents to react with aldehyde groups
resulting in deep pink color
Feulgen rxn
periodic acid cleaves bonds between adjacent carbons of carbohydrates, thus forming aldehyde groups, which allows _______. what is this rxn called?
Schiff reagents can react with aldehyde groups
resulting in deep pink color
PAS = periodic acid-Schiff rxn
substances that can be displayed via PAS
polysaccharides glycosaminoglycans proteoglycans glycoproteins glycolipids
dye used to show glycogen deposits of the ____ .
best carmine
deposits in the liver
what stains are used for RNA staining?
basic dyes
in RNA staining ____ slides are needed for …..? what are these slides incubated with?
control slides — to distinguish between other basophilic substances
incubated with ribonuclease
used to study structure and function of macromolecules within a specimen
cytochemistry
agents used in cytochemistry
Schiff reagent rxn
Feulgen rxn
PAS
best carmine
what does PAS stand for?
periodic acid Schiff rxn
