Using a Bright Field Light Microscope

1
Using a Bright Field Light Microscope

• David R. Caprette, Ph.D.

2
Mounting a Specimen
Start with lowest power Specimen side UP
Mechanical stage
Spring clip
YES
Slide
Specimen up
Opening in stage
4x
Slide
Translational controls
NO!
Specimen down
Forward/back
4x
Slide
Left/right
3
Adjusting the Oculars
Focusing an eyepiece (twist to change focus)
Eye separation
center
Closed
Wide open
Double image
Twist to extend eyepiece tube
Adjusted for a single image
4
The Condenser
Top view of condenser with selector turret
Condenser aperture three positions
Fully stopped down
Optimized for contrast and resolution
Side view, condenser lowered
Wide open
5
Find the Target at Low Power
Find the target and adjust contrast.
(left) Low contrast image is difficult to detect
if somewhat out of focus. (right) With aperture
diaphragm stopped down, image is visible although
distorted.
If you cannot find the target, start by focusing
on an artifact in the focal plane of the specimen.
A air bubble B (w/arrow) coverslip
edge C scratch on slide surface
All images 100x
A
B
C
6
Strategy for Working up in Magnification
A
B
C
Move slide down to move image up.
Move slide up to move image down.
Move slide left to move image right.
Move slide right to move image left.
Focused image at 40x target circled
Centered and focused at 40x
At 100x, target is off center and out of focus.
D
E
F
Move slide right to move image left
Centered and focused at 100x
At 400x, target is again off center and out of
focus.
Centered and focused at 400x
7
Focusing at High Magnification
40x objective positioned over upside down slide
10x
correct position
10x
too close
Objective fully compressed
Specimen down
Oops! ...a 300 mistake!
40x
40x
normal position
contact with slide
8
Focusing on Multiple Surfaces
Objective lens
Drawing not to scale
Initial focus above slide
Slide
Condenser lowered for clarity
Focusing direction
9
Looking Through a Specimen
Focusing through a Spirogyra filament (400x)
raise stage
raise stage
Chloroplasts in focus just beneath cell wall.
Approaching middle of the cell.
Top of cell in focus.
raise stage
raise stage
raise stage
Focused at the center.
Approaching the bottom.
Chloroplasts in focus just above cell wall.
10
Oil Immersion Microscopy
Dry magnification
Oil immersion
Diffraction severely compromises resolution.
Diffraction is minimized with oil interface.
A
B
B Bacteria at 1000x with oil immersion objective
lens (center portion of field in A)
A Bacteria at 400x dry magnification, showing
melted appearance (poor resolution)
11
Placing an Oil Immersion Lens
Focus at high dry magnification.
Move the high dry lens out of the way.
Place an oil drop over the specimen.
CAREFULLY place the oil immersion lens.
Place oil directly on a coverslip
Place oil directly on a smear no coverslip
Lens tip must be completely immersed.
Ready for viewing
12
Focusing with an Oil Immersion Lens
Too close move stage down
Good some focusing needed
Too far away move stage up
high just right
low
No visible image
No visible image
13
Dirty Lenses?
If you see marks against an empty field, move the
slide from left to right.
If the marks arent on the slide, rotate the
ocular.
Out of focus spots may be on the condenser lens.
If so...

...lowering the condenser will take them further
out of focus.
...movement indicates spots are on the slide.
...movement indicates spots are on the ocular
lens.