Brief history of the light microscope Janssen and Janssen (1590): the first light microscope: 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 MICROSCOPE

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

Ernst Abbe’s formula: D=0.6x /Nxsin  where D: resolution (min. distance between two points sensed as two separate entities)  the wavelength of light (0.53) N: refraction index (=1 in vacuum and air, =1,6 in immersion oil) and air, =1,6 in immersion oil)  the aperture angle of the lens

RESOLUTION VALUES: Human eye: 0.2 mm Best light microscope: 0.2  m (x1000 useful magnification) Electron microscope: SEM:1.0 nm (x useful magnification) TEM:0.1 nm (x useful magnification)

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

CLASS LIGHT MICROSCOPE Controls I 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 left rear

CLASS LIGHT MICROSCOPE Controls II Base Condenser Eyepiec e/Ocular Slide Light Body Stage clip for slide Condenser focusing Condenser centering Ocular focusing left- side

OPERATION I 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 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 90 o 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

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

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

50 % ethanol 70 % ethanol 95 % ethanol 100 % ethanol benzene/ xylene Dehydrating series paraffin wax Remove the water & replace with wax-solvent Embed the oriented specimen in molten wax Miscible with ethanol; dissolves wax Fresh tissue 10% Formalin fixative label

MICROTOME - a fancy meat- slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically Block Knife Section Glass slide Water-bath After it is solid, hold the wax block & cut slices Mount the thin slices (sections) on slides Lift out floating section on the slide

FREEZING MICROTOME holds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically Block is the tissue Knife Section Water-bath Glass slide For fast biopsy, embedding is omitted - frozen sections Mount the thin slices (sections) on slides Lift out section on the slide

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

SOME EXAMPLES OF HEMATOXILIN-EOSIN STAINING Seromucous gland Serous gland

SOME OTHER OFTEN USED STAINING METHODS Alcian blueAzanResorcin-fuchsin SchmorlGiemsa Silver-impregnation SchmorlGiemsa Silver-impregnation

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

Images versus REALITY Artifacts are appearances not true to the original state of the tissue SLIDE PREPARATION Artifacts Excise & Fix (preserve) the tissue in fixative 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 Knife scores, chatter Bruising/splitting from cutting; Poor preservation, e.g., gut lining, enzymes, lost fat Wrinkles, section not flat, splits Weak/unbalanced staining Dirt, hair, bubbles Dirt on lenses, bad illumination Misleading orientation, Shrinkage & distortion, Mislabeled

Some differences between light and electron microscopy I LIGHT MICROSCOPY ELECTRON MICROSCOPY Section thickness (1-30  m) 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.

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  m. 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.