3.10.3 Tips For Diascopic Fluorescence Microscopy - Nikon ECLIPSE Si Instructions Manual

3.10.3 Tips for Diascopic Fluorescence Microscopy

Diascopic fluorescence microscopy is a microscopy method for using diascopic illumination to examine specimens
stained with fluorescent dye or fluorescent protein. You can observe excited fluorescence as an image by directing
diascopic illumination of a particular wavelength at objects stained with special fluorescent dye.
Purpose of diascopic fluorescence microscopy
This microscope can be used for observation by diascopic fluorescence (GFP-B) by using the E2-F-Fl diascopic
fluorescence filter set (GFP-B). You can perform observation using GFP, FITC, Alexa 488, and more.
Note on the brightness of excited fluorescence
The brightness of fluorescent light excited during diascopic fluorescence microscopy might be reduced, for
example due to the expression efficiency of the sample GFP.
Generally, excited fluorescent light is extremely faint.
When performing fluorescence microscopy, darken the area around the microscope, for example by turning
down the ambient light so that you can observe the specimen correctly.
Note on eye protection during slider operation
Move your eyes away from the binoculars during position switching of EX filter sliders.
Fluorescence microscopy is performed with diascopic illumination set to maximum. Therefore, bright light
might reach the binoculars when the position of the fluorescence microscopy slider is switched.
Do not remove the slider.
Never remove the EX filter slider from the microscope during diascopic fluorescence microscopy.
Preventing UV light (using a shielding plate)
Use the shielding plate to prevent strong light including UV light from the specimen through the condenser
from entering your eyes.
Protecting specimens and preventing discoloration
Brightly illuminating a specimen for fluorescence microscopy for an extended time might cause damage or
discoloration of the specimen.
When stopping diascopic fluorescence microscopy, either turn off the illumination or lower the brightness.
Make this a habitual practice.
Using non-fluorescent glass slide, a cover glass, and immersion oil
To obtain an image with good contrast during fluorescence microscopy, make sure that you use
non-fluorescent glass slide, a cover glass, and an immersion oil specified by our company.
Limiting illumination to the field of observation (adjusting the field diaphragm)
"Adjusting the field diaphragm" means narrowing the illumination to limit the field of observation of the
specimen.
Usually, observation is performed with the field diaphragm narrowed until it roughly circumscribes or inscribes
the edge of the field of view. If the field diaphragm is opened too wide, causing an unnecessarily wide field of
illumination, stray light enters from the outside, resulting in flares and reducing the contrast of the optical
image. This also causes the specimen's discoloration field to become too wide.
Each time you change the objective, adjust the field diaphragm.
Finding targets inside the specimen
For fluorescence microscopy, the general practice is to first use phase difference to find a target and then
switch to using fluorescence microscopy. However, when finding targets using bright-field microscopy, you will
need to take the following kinds of measures:
• In bright-field microscopy, first use a 10x objective, and then narrow the condenser as required.
• If raising the magnification makes it difficult to find the target, switch to fluorescence microscopy, and then
find the target while suppressing the excitation light.
Another method is to work while looking at the edge plane of the cover glass to identify the rough position of
the target.
Chapter 3 Detailed Explanation
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