1. Main instrument of
histology:
the light microscope
The

2. 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: micro=small,
scopeo= observe: the name
MICROSCOPE

3. Marcello Malpighi (XVIIth century): the first scientist
using the microscope for scientific research

4. Ernst Abbe’s formula:
D=0.6xl/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

5. RESOLUTION VALUES:
Human eye: mm
Best light microscope: 0.2 mm
(x1000 useful magnification)
Electron microscope:
SEM: nm
(x useful magnification)
TEM: nm
(x useful magnification)

6. CLASS LIGHT MICROSCOPE
Eyepiece/Ocular
Objective lenses
Max MAGNIFICATION
Eyepiece (10X) times ‘Oil’ Objective (100X) = 1000X
Stage
Slide
Body
Condenser
Base
Light source

7. 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

8. CLASS LIGHT MICROSCOPE Controls II
Ocular focusing
Eyepiece/Ocular
Base
Stage clip for slide
Condenser focusing
left-side
Slide
Body
Condenser
Condensercentering
Light

9. Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Field diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
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

10. Base
Condenser
Eyepiece/Ocular
Slide
Light
Body
Inter-ocular distance
Moving stage
Iris diaphragm
Coarse & Fine focus
Light intensity
On/Off
Objective selection
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

11. 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

12. Collection of samples Fixation
From dead bodies (as quickly as possible after death)
Smears from bodily fluids (eg. blood)
From biopsies (eg. liver, large intestine, etc.)
From cell cultures (maybe alive)
From experimental animals after fixation (rat, mouse, etc.)
Fixation
Dehydrating agents (ethanol, acetone, etc.)
Salts of heavy metals
Aldehydes (formaldehyde)

13. 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
Fresh tissue
10% Formalin fixative
benzene/xylene
Miscible with ethanol; dissolves wax
paraffinwax

14. 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

15. Mount the thin slices (sections) on slides
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

16. Stain with Hematoxylin - blue
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”

17. SOME EXAMPLES OF HEMATOXILIN-EOSIN STAINING
Seromucous gland
Serous gland

18. SOME OTHER OFTEN USED STAINING METHODS
Alcian blue Azan Resorcin-fuchsin
Schmorl Giemsa Silver-impregnation

19. MICROSCOPIC SLIDE Side view of slide
Glass coverslip
Tissue Section
Mounting medium
Label
Glass slide 1”X3”
Mounting medium: permeates section; fastens coverslip to slide; is clear; has refractive index as for glass

20. 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
Label ~
The label may have been put on the non-coverslip side, as shown

21. 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

22. 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.

23. 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
orientation 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.