The

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Main instrument of histology the light microscope
The
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Brief history of the light microscope Janssen
and Janssen (1590) the first light
microscope 2 m long copper tube. Magnification
x60
Galileo Galilei (1610) small handy table
microscope Faber of Bamberg microsmall, scope
o observe the name MICROSCOPE
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Marcello Malpighi (XVIIth century) the first
scientist using the microscope for scientific
research
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Ernst Abbes formula D0.6xl/Nxsina
where D resolution (min. distance between two
points sensed as two separate entities) l the
wavelength of light (0.53) N refraction index
(1 in vacuum and air, 1,6 in immersion
oil) a the aperture angle of the lens
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RESOLUTION VALUES Human eye 0.2 mm Best
light microscope 0.2 mm (x1000 useful
magnification) Electron microscope SEM 1.0
nm (x400.000 useful magnification) TEM 0
.1 nm (x1.000.000 useful magnification)
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CLASS LIGHT MICROSCOPE
Eyepiece/Ocular
Objective lenses
Max MAGNIFICATION Eyepiece (10X)
times Oil Objective
(100X) 1000X
Stage
Slide
Body
Condenser
Base
Light source
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CLASS LIGHT MICROSCOPE Controls I
Eyepiece/Ocular
Inter-ocular distance
Objective selection
Iris diaphragm
Slide
Body
Coarse Fine focus
Condenser
Moving stage
Light intensity
Base
Field diaphragm
Light
On/Off
left rear
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CLASS LIGHT MICROSCOPE Controls II
Ocular focusing
Eyepiece/Ocular
Stage clip for slide
Condenser focusing
left-side
Slide
Body
Condenser
Condensercentering
Light
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OPERATION I
Without looking down the eyepieces, plug in the
cord Turn the light-intensity
knob back counterclockwise, Switch
on the light, turn the intensity up (about a 90o
turn) while observing the
light via the field opening
Open the field diaphragm wide
Move the
condenser assembly to its top position
Switch the shortest objective lens (X4)
into the working position Open the iris
diaphragm wide
Select any well-stained
slide
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OPERATION II
Field diaphragm
Pull back the clip place slide, cover-slip up,
on the stage Use the stage controls to
bring the stained section over the light Focus,
using coarse, then fine adjustments
Close the iris diaphragm to
take the glare out of the view Push (pull)
the eyepieces together to match your eye spacing
Shut one eye, focus with the fine focus
then shut that eye, open the other, and focus for
it with the ocular focus (turning the eyepiece
knurled ring)
Switch in the next
higher objective, and focus, using the main
focusing controls testing for binocular fusion
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SLIDE PREPARATION for light microscopy
Excise Fix (preserve) the tissue in fixative
Remove the water replace with wax-solvent
Embed the oriented specimen in molten wax
After it is solid, hold the wax block cut
slices
Mount the thin slices (sections) on slides
When dry, remove the wax, stain the section
Remove surplus stain water mount coverslip
When mounting medium has set, do microscopy
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Remove the water replace with wax-solvent
Embed the oriented specimen in molten wax
50 ethanol
70 ethanol
95 ethanol
Dehydrating series
label
100 ethanol
benzene/xylene
paraffinwax
Miscible with ethanol dissolves wax
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After it is solid, hold the wax block cut
slices
Knife
Section
Block
Glass slide
MICROTOME - a fancy meat-slicer - holds the wax
block, cuts off thin slices, as the block is
slowly advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out floating section on the slide
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For fast biopsy, embedding is omitted - frozen
sections
Knife
Section
Block is the tissue
Glass slide
FREEZING MICROTOME holds the frozen tissue,
cuts off thin slices, as the block is slowly
advanced mechanically
Water-bath
Mount the thin slices (sections) on slides
Lift out section on the slide
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When dry, remove the wax, stain the section
Dissolve paraffin wax
Stain with Hematoxylin - blue
Wash
Potassium eosinate- stain charged
amine, etc, groups on proteins bind -eosin
Acidophilic staining
Stain with eosin - red
Wash
Nuclei - blue Cytoplasm- red
Basophilic
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SOME OTHER OFTEN USED STAINING METHODS
Alcian blue Azan Resorcin-fuchsin
Schmorl Giemsa Silver-impregnation
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MICROSCOPIC SLIDE Side view of slide
Glass coverslip
Tissue Section
Mounting medium
Label
Glass slide 1X3
Mounting medium permeates section
fastens coverslip to slide is clear
has refractive
index as for glass
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SLIDE USE - Cautions
GLASS IS FRAGILE !
Take care with individual slides especially
with the boxes of slides
The slide must go on the stage coverslip up
The high-dry oil objectives cannot focus
through the thickness of the slide to the section
The label may have been put on the non-coverslip
side, as shown
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SLIDE PREPARATION Artifacts
Images versus REALITY Artifacts are
appearances not true to the original state
of the tissue
Excise Fix (preserve) the tissue in fixative
Bruising/splitting from cutting Poor
preservation, e.g., gut lining, enzymes, lost
fat
Embed the oriented specimen in molten wax
Misleading orientation, Shrinkage
distortion, Mislabeled
After it is solid, hold the wax block cut
slices
Knife scores, chatter
Mount the thin slices (sections) on slides
Wrinkles, section not flat, splits
When dry, remove the wax, stain the section
Weak/unbalanced staining
Remove surplus stain water mount coverslip
Dirt, hair, bubbles
When mounting medium has set, do microscopy
Dirt on lenses, bad illumination
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Some differences between light and electron
microscopy I LIGHT MICROSCOPY
ELECTRON
MICROSCOPY ---------------------------------------
--------------------------------------------------
------------------------------ Section thickness
(1-30 mm) gives Very thin
sections provide no a little depth of focus for
depth of focus,
but 3-D information appreciation of the third
dimension. can be had from (a)
thicker sections Serial sections can be cut,
viewed by high-voltage EM (b)
shadowed and used to build a composite image
replicas of fractured surfaces (c) or
representation.
scanning electron microscopy
(SEM). Most materials and structures cannot
Heavy metal staining gives a more be
stained and viewed at the same
comprehensive picture of membranes, time stains
are used selectively to
granules, filaments, crystals, etc. give a
partial picture, e.g. a stain
but this view is incomplete and even for mucus
counterstained to show visible
bodies can be improved by cell nuclei.

varying the technique. Specimen can be large and
Specimen is in
vacuo. Its small size even alive.
creates
more problems with sampling

and orientation.
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Some differences between light and electron
microscopy II LIGHT MICROSCOPY
ELECTRON
MICROSCOPY ---------------------------------------
--------------------------------------------------
---------------------------- Image is presented
directly to the Image is in
shades of green on eye. Image keeps the colours
given the screen
photographically, the specimen by staining.
only in black and
white. Modest magnification to X 1500
High magnification,up to X
2,000,000 but a wider field of view and easier
thus the range of magnification orien
tation
is greater
Resolving power to 0.25 mm.
Resolving power to 1 nm (0.001
mm.) Frozen sections can yield an image
Processing of tissue takes a day at within
20 minutes.
least. Crude techniques of preparation
High resolution and
magnification introduce many artefacts.
demand good fixation (e.g.
by (Histochemical methods are better.)
vascular perfusion), cleanliness

and careful cutting, adding up to

fewer artefacts.