brief history of the light microscope janssen and janssen (1590): the first light microscope: the...
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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.