Digital Microscopy Center, University of Washington
July 29, 2016
5
A. Performing Brightfield transmitted light microscopy
1.
Focus on specimen
1.1.
Place slide on the stage and hold in place with the stage clip
1.1.1.
Begin with low magnification for greater ease in surveying your specimen to
identify regions of interest
2.
Adjust the focusing eyepiece
2.1.
Find a small object with sharp boundaries
2.2.
Use the fine focus using observing with one eye
2.3.
rotate the upper portion of the other eye piece until the object is in similar focus with both
eyes
3.
Set Koehler Illumination for objectives 10X and above (Figure 1, Figure 2)
3.1.
Note: this should be performed every time the objective is changed
3.2.
Focuses the condenser to the same plane as the objective, set the incoming light column and
condenser NA
3.2.1.
This is essential for best contrast, resolution and even illumination
3.3.
Open the condenser diaphragm for maximum brightness
3.4.
Make certain the condenser top lens is flipped up
3.5.
Close the field stop diaphragm to its minimum diameter
3.6.
Use the condensor focus knob to bring the leaves of the field stop sharply into focus while
observing through the eyepieces
3.7.
Roughly center the spot of light with the centering screws
3.8.
Open the field stop until it just reaches the edge of the field of view
3.9.
Adjust the centering until it is concentric with the field of view
3.10.
Close the condenser diaphragm to match the NA of the objective
3.10.1.
If the objective NA is higher than .85, set condenser to maximum
3.11.
Double check the eyepieces – if the image is blurry, close the field stop, re-focus condenser
then re-open the field stop
4.
Set Koehler Illumination for objectives below 10X
4.1.
Follow the steps above, except that after step 3.7, flip the condenser top lens out of position
5.
Setting contrast filters
5.1.
A color filter inserted into the light path, as paddles at the lamp housing or placed on the
condenser, can increase contrast.
5.1.1.
Viewing blue or brown staining often benefits from using a green or blue filter
