The

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: 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)

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

9. 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 OPERATION I Without looking down the eyepieces, plug in the cord Turn thelight-intensity knob back counterclockwise,Switch on the light,turn the intensity up (about a 90o turn)while observing the lightvia the field openingOpen the field diaphragm wideMove thecondenser 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. Eyepiece/Ocular Inter-ocular distance Objective selection Iris diaphragm Slide Body Coarse & Fine focus Condenser Moving stage Light intensity Base Light On/Off OPERATION II Field diaphragm Pull back the clip & place slide, cover-slip up, on the stageUse the stage controls to bring the stained section over the lightFocus, using coarse, then fine adjustmentsClose 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. Fresh tissue 10% Formalin fixative Remove the water & replace with wax-solvent Embed the oriented specimen in molten wax 50 % ethanol 70 % ethanol 95 % ethanol label Dehydrating series 100 % ethanol benzene/xylene paraffinwax Miscible with ethanol; dissolves wax

13. 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

14. 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

15. When dry, remove the wax, & stain the section Dissolve paraffin wax Stain with Hematoxylin - blue Wash Potassium+eosinate- stain+ charged amine, etc, groupson proteins bind-eosin “Acidophilic staining” Stain with eosin - red Wash Nuclei - blue Cytoplasm- red “Basophilic”

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

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

18. 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

19. Label ~ 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

20. SLIDE PREPARATION Artifacts ImagesversusREALITYArtifacts 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 Weak/unbalanced staining When dry, remove the wax, & stain the section Dirt, hair, bubbles Remove surplus stain & water; mount coverslip When mounting medium has set, do microscopy Dirt on lenses, bad illumination

21. 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.

22. 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.