Transcript No Slide Title

The
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
Marcello Malpighi (XVIIth century): the first scientist
using the microscope for scientific research
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
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)
CLASS LIGHT MICROSCOPE
Eyepiece
/Ocular
Objective lenses
Max MAGNIFICATION
Stage
Slide
Body
Condenser
Eyepiece (10X)
times
‘Oil’ Objective (100X)
= 1000X
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
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 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
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
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
70 %
ethanol
95 %
ethanol
Fresh
tissue
10% Formalin
fixative
label
100 %
ethanol
benzene/
xylene
Miscible with ethanol; paraffin
dissolves wax
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
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
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 2,000,000
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.