Methods Microscopy

8/6/2019 Methods Microscopy

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MICROSCOPIC ANATOMYMICROSCOPIC ANATOMY


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All living organisms are constructed from cells.

Cells come in varied shapes and sizes


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Interaction of probe used (photons: light,phase contrast, polarizing & fluorescence

microscopy; electron beams: EM), and tissue

components produce image

Considerations in microscopic analysis:that the probe being utilized must not be

larger than the detail to be seen

that the probe and object being

investigated must interactit must be possible to observe and

interpret this interaction

Units for measuring microscopic dimensions:

MICROSCOPY


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WHAT

CAN

WE

SEE?


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Magnification increases the apparent size

of the specimen; a property of both ocular &

objective lenses

Working distance is the distance between the

specimen and the magnifying lens.

Depth of field is a measure of the amount of

a specimen that can be in focus. Highly

sensitive video cameras enhance power of

microscopes, and create digitized images

that can be fed into computers for

quantitative image analysis

IMPORTANT TERMS IN MICROSCOPY


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Numerical aperture a measure of the size or

angle of the cone of light delivered by the

illuminating condenser lens to the object planeand of the cone of light emerging from the object.

Resolving power a measure of linear distance of

the smallest degree of separation at which 2

details can still be distinguished from each other;dependent on quality of objective lens; R also

varies according to the refractive index at the

interface of the media used

Generally resolution increases withmagnification, although there comes a point of

diminishing returns where magnification is

increased beyond added resolution gain.


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1.Fixation - prompt treatment of tissues infixatives

for about12 hrs. (depending on tissue size)

prevent autolysis by enzymes or bacteria, and

preserve their morphologic and molecular

composition.

To render the structural components insoluble,

chemicals that precipitate the proteins are used.

The best fixatives are those that produce fine

precipitates, e.g. buffered isotonic solution of 4%formaldehyde and glutaraldehyde react with

amine groups (NH2) of proteins, or cross-link

with protein

PREPARATION OF TISSUES FOR MICROSCOPIC

EXAMINATION (MICROTECHNIQUE)


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Gross distortions without basis in the structure

of the living cell are termedfixation artifacts.

Examples of artifacts: swelling and shrinkage of tissue

components due to poor fixation,

dehydration and/or embedding techniques;

wrinkles, tears, air bubbles due to poor

sectioning technique;

dust and stain precipitate in section

resulting from use of old stain solutions, use

of improperly filtered or unfiltered stain

solutions, mistakes made during

preparation of the stain, or poor staining

technique.


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Folded artifact Cracked tissue artifact

Knife mark + folded artifact


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2.Dehydration and Clearing

Bathing of tissue in graded concentrations of

organic solvents (70-100% ethanol) to replace

tissue water within 6-24 hrs.

Ethanol is then replaced with solvent miscible

in embedding medium (xylene, benzene,

toluene) WHY? Most fixatives are water soluble, most

embedding media are non-polar and are not

miscible with water.

Dehydration moves the tissue from a polar(water-based) medium to a non-polar medium

(e.g. toluene) that is miscible with the

embedding medium.


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3.Embedding in melted paraffin at 58-600C, or

plastic resin at room temperature

Tissue will be sectioned, and needs to be

durable enough to withstand the sectioning

process.

Embedding in wax or plastic immobilizes

structural components of tissue. Holds themin place as sectioning is done.

Embedding medium must penetrate all

cellular/intercellular spaces to impart rigid

consistency to tissue before sectioning Tissue shrinkage and artifacts may result

from heat needed for paraffin embedding;

virtually absent in resin embedding


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4.Sectioning by

microtome to a

thickness of1-10 Qm Sections are then

floated in warm water

and transferred to

glass slides Allows histologist to:

see internal

structure of tissue.

stains or specific

markers such as antibodies to more easily

infiltrate the tissues.

light to pass through tissue making structure

visible.


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Images from thin sections

are 2-D; living tissues are 3-D

In order to understand the

architecture of an organ,

sections made in different

planes should be studied

How different3-dimensional

structures may appear when

thin-sectioned. A: Different

sections through a hollow

ball and a hollow tube. B: A

section through a single

coiled tube may appear as

sections of many separate

tubes. C: Sections through a

solid ball (above) and

sections through a solid

cylinder (below).


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5.Staining to differentiate the

colorless tissue elements as

certain cellular elementstake up more stain than

others, producing a contrast

that allows observation of

structure not visible in

unstained tissue. It may alsoreveal differences in

chemical nature of regions

of the tissue.

6.Mounting stained sections

are placed on a slide in a

gummy medium that

hardens. The preparation is

then covered with a thin

cover glass.


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1.Light microscopes-

compound, dissecting,

brightfield, and phase-

contrast Best resolution is 0.2

m.

Maximum

magnifications are

between 1000X and

1250X.

Anton vanLeeuwenhook

(1632-1723)

TYPES OF

MICROSCOPES


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COMPOUND MICROSCOPE DISSECTING MICROSCOPE


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Compound

microscopes

bring small

objects "closer"

to the observer

by increasing the

magnification of

the sample. Since the sample

is the same

distance from the

viewer, a "virtual

image" is formed

as the light

passes through

the magnifying

lenses.


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Bright-field

Nomarski differential-

phase-contrast

phase-contrast

dark-field

Phase contrast microcopy- uses a lens system that changes

light speed as it passes through structures with different

refractive indices The phase of the light is altered by its passage through

the cell, and small phase differences can be made

visible by exploiting interference effects

Phase-contrast and differential interference optics

produce 3-D images of transparent living cells, tissues


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Contrast in

LightMicroscopy

Contrast inPhase

Microscopy


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2.Fluorescence microscopy uses strong UV light

source that irradiate substances dyed with

fluorescent stains, e.g.

acridine-orange

These appear as

brilliant, shiny particles

on a dark background;

useful for identifying& localizing NA in cells

Fluorescence spectros-

copy analyzes light

emitted by fluorescent

compounds in a micro-spectrophotometer

This permits highly

sensitive assays of

cellular substances such as catecholamines


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Theconfocal

microscope

produces

opticalsections by

excluding

out-of-focus

light


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3.Polarizing

microscopy

birefrigentsubstances rotate

direction of

polarized light

emerging frompolarizing filters

Useful for

visualizing

substances withrepetitive, oriented

molecular

structuresCollagen fibers,

polarizing microscopy

Compact

bone


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4.Electron microscopy uses high energy electron

beams (between 5,000 - 109 electron volts)

focused through electromagnetic lenses. Interaction of electrons deflected by lenses

beamed on tissue components permits high

resolution (0.2 - 1 nm) and 400x greater

magnification than light microscopes The increased resolution results from the shorter

wavelength of the electron beam

Disadvantages of EM: requirements of a vacuum-

enclosed system, high voltage, mechanicalstability; special treatment & sample preparation

make it highly complex and costly; requires the

services of well-trained personnel


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TE

M

S

E

M


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Scanning vs. Transmission EM:

In the TEM, the image is formed directly on the image

plane.

In the SEM, the image is formed indirectly by

accumulation of information from the specimen point

by point.

There is no need to cut ultra thin sections becausethe beam of the SEM does not pass through the

specimen.

The resolution of the SEM is about100 Angstrom vs.

4-5 Angstrom achieved by the transmission type.

The SEM has great depth of field making it possibleto obtain 3-D images.

TEM magnifications are commonly over 100,000X

SEM displays images on high resolution TV monitors.


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SEM: T-lymphocyte, E.coli

attacked by macrophage

TEM: mitochondria &

chloroplast


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Specimen Preparation for EM:

Fixation in osmium tetroxide, osmium dichromate,

acrolein and glutaldehyde. Since registration of color is not possible with the

EM system, staining with colored dyes is not done in

EM studies.

Specimen is mounted on a copper grid covered withcarbon and/or plastic film


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Freeze-cleaving, Freeze-

etching or Cryofracture

methods

Used with EM; replicas are

made of surfaces of frozen

aqueous materials at very

low temperatures in vacuo The use of chemical

fixatives, dehydrating and

embedding agents are

avoided by using a freezing

microtome/cryostat whichpermit sections to be

obtained without

embedding


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chloroplast thylakoid membranes

Freezing does not inactivate

most enzymes, hinders

diffusion of smallmolecules, eliminates

dissolution of tissue lipids

by solvents

The tissue is impregnated

with a 25% glycerolsolution

before rapidfreezingin

liquidnitrogenorFreon12 at

1000C to1550C.

Notentirelyfree ofartifacts;valuable inthe studyof

membranes andtheir

junctionalspecializations.

vesicles


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QUESTIONS?


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REFERENCES

Bloom and Fawcett. 12th ed. Textbook ofHistology

de Fiore, Atlas ofHistology

Junquiera, LC and Carneiro, J. Basic Histology: Text and Atlas, 12th ed

Ross, M. et. al., 4th ed. BasicHistology: Text and Atlas

Kuehnel,W. Color Atlas of Cytology, Histology and Microscopic

Anatomy

Young, B.Wheaters FunctionalHistology: A Text and Color Atlas

http://www.northland.cc.mn.us/biology/AP2Online/Nervous/Default.htm

http://highered.mcgraw-

hill.com/sites/0072495855/student_view0/histology_atlas.html

http://www.meddean.luc.edu/lumen/meded/Histo/frames/histo_frames.html

http://projects.galter.northwestern.edu/rhodin/

http://www.siumed.edu/~dking2/index.htm

http://www.bu.edu/histology/m/i_main00.htm

http://www.histology-world.com/

http://www.mhhe.com/biosci/ap/histology_mh/start_histology.html

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Methods Microscopy