The Brief history of the light microscope Janssen

The

Brief history of the light microscope Janssen and Janssen (1590): the first light microscope: 2 m long copper tube. Magnification x 60 Galileo Galilei (1610): small handy table microscope Faber of Bamberg: micro=small, scopeo= observe: the name MICROSCOPE

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

Ernst Abbe’s formula: D=0. 6 xl/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

RESOLUTION VALUES: Human eye: 0. 2 mm Best light microscope: 0. 2 mm (x 1000 useful magnification) Electron microscope: SEM: 1. 0 nm (x 400. 000 useful magnification) TEM: 0. 1 nm (x 1. 000 useful magnification)

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

CLASS LIGHT MICROSCOPE Controls I Eyepiece/ Ocular Inter-ocular distance Objective selection Iris diaphragm Slide Body Coarse & Fine focus Condenser Moving stage Field diaphragm Light intensity Base Light On/Off left rear

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

OPERATION I Eyepiece/ Ocular Inter-ocular distance Objective selection Iris diaphragm Slide Body Coarse & Fine focus Condenser Moving stage Field diaphragm Light intensity Base Light On/Off 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 (X 4) into the working position Open the iris diaphragm wide Select any well-stained slide

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

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

Remove the water & replace with wax-solvent Embed the oriented specimen in molten wax 50 % ethanol De 70 % ethanol hy dra 95 % ethanol tin gs eri Fresh tissue 10% Formalin fixative es label 100 % ethanol benzene/ xylene Miscible with ethanol; dissolves wax paraffin wax

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

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

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” Nuclei - blue “Basophilic” Cytoplasm- red Stain with eosin - red Wash

SOME EXAMPLES OF HEMATOXILIN-EOSIN STAINING Seromucous gland Serous gland

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

MICROSCOPIC SLIDE Side view of slide Glass coverslip Mounting medium Label Glass slide Tissue Section 1”X 3” Mounting medium: permeates section; fastens coverslip to slide; is clear; has refractive index as for glass

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

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 Mount the thin slices (sections) on slides Knife scores, chatter Wrinkles, section not flat, splits When dry, remove the wax, & stain the section Remove surplus stain & water; mount coverslip When mounting medium has set, do microscopy Weak/unbalanced staining Dirt, hair, bubbles Dirt on lenses, bad illumination

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 be stained and viewed at the same time; stains are used selectively to give a partial picture, e. g. a stain for mucus counterstained to show cell nuclei. Heavy metal staining gives a more comprehensive picture of membranes, granules, filaments, crystals, etc. ; but this view is incomplete and even visible bodies can be improved by varying the technique. Specimen can be large and even alive. Specimen is in vacuo. Its small size 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 eye. Image keeps the colours given the specimen by staining. Image is in shades of green on the screen; photographically, only in black and white. Modest magnification to X 1500; but a wider field of view and easier orientation High magnification, up to X 2, 000 thus the range of magnification is greater Resolving power to 0. 25 mm. Resolving power to 1 nm (0. 001 mm. ) Frozen sections can yield an image within 20 minutes. Processing of tissue takes a day at least. Crude techniques of preparation introduce many artefacts. (Histochemical methods are better. ) High resolution and magnification demand good fixation (e. g. by vascular perfusion), cleanliness and careful cutting, adding up to fewer artefacts.