TheThe

Brief history of the light microscopeBrief history of the light microscope

Janssen and Janssen (1590):Janssen and Janssen (1590): the first light microscope:the first light microscope:2 m long copper tube.2 m long copper tube.Magnification x60Magnification x60

Galileo Galilei (1610): small handy Galileo Galilei (1610): small handy table microscopetable microscope

Faber of Bamberg: micro=small,Faber of Bamberg: micro=small,scopeo= observe: the namescopeo= observe: the name

MICROSCOPEMICROSCOPE

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

Ernst Abbe’s formula:Ernst Abbe’s formula:

DD==0.60.6xx//NNxxsinsinwherewhere

D: resolution (min. distance betweenD: resolution (min. distance betweentwo points sensed as two separate two points sensed as two separate entities)entities)the wavelength of light (0.53)the wavelength of light (0.53)N: refraction index (=1 in vacuumN: refraction index (=1 in vacuum and air, =1,6 in immersion oil)and air, =1,6 in immersion oil)the aperture angle of the lensthe aperture angle of the lens

RESOLUTION VALUES:RESOLUTION VALUES:

Human eye: Human eye: 0.2 mm0.2 mm

Best light microscope: Best light microscope: 0.2 0.2 m m (x1000 useful magnification)(x1000 useful magnification)

Electron microscope:Electron microscope:SEM:SEM: 1.0 nm1.0 nm

(x400.000 useful magnification)(x400.000 useful magnification)TEM:TEM: 0.1 nm0.1 nm

(x1.000.000 useful magnification)(x1.000.000 useful magnification)

CLASS LIGHT MICROSCOPE

Max MAGNIFICATION

Eyepiece (10X) times ‘Oil’ Objective

(100X) = 1000XBase

Eyepiece/Ocular

StageSlide

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

Eyepiece/Ocular

Slide

Light

Body

Stage clip for slide Condenser

focusing

Condensercentering

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 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

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 fixativeRemove 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 %

ethanol95 % ethanol

100 % ethanol

benzene/xylene

Dehydrating series

paraffinwax

Remove the water & replace with wax-solventEmbed 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 METHODSSOME OTHER OFTEN USED STAINING METHODS

Alcian blueAlcian blue AzanAzan Resorcin-fuchsinResorcin-fuchsin

SchmorlSchmorl GiemsaGiemsa Silver-impregnation 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 noa little depth of focus for depth of focus, but 3-D informationappreciation of the third dimension. can be had from: (a) thicker sectionsSerial sections can be cut, viewed by high-voltage EM; (b) shadowedand 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 morebe 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 evenfor mucus counterstained to show visible bodies can be improved bycell nuclei. varying the technique.

Specimen can be large and Specimen is in vacuo. Its small sizeeven 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 oneye. 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 2,000,000but a wider field of view and easier thus the range of magnificationorientation 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 atwithin 20 minutes. least.

Crude techniques of preparation High resolution and magnificationintroduce many artefacts. demand good fixation (e.g. by(Histochemical methods are better.) vascular perfusion), cleanliness and careful cutting, adding up to fewer artefacts.