Diascopic Fluorescence Microscopy - Nikon ECLIPSE Si Instructions Manual

2.4

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.
This microscope can be used for observation by diascopic fluorescence (GFP-B) by using the E2-F-FL Dia-FL
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.
Nikon recommends darkening the room because images are not clearly visible if the ambient light is too
bright.
For details on the configuration and the assembly of the microscope for diascopic fluorescence microscopy,
see "4.2.4 Configuration for Diascopic Fluorescence Microscopy."
1 Prepare the microscope.
Attach the following optical parts and the shielding
plate to the microscope in advance:
(1) EX filter slider
When performing bright-field microscopy, bring the
BF position into the optical path, and when
performing diascopic fluorescence microscopy, bring
the GFP position into the optical path.
(2) BA filter slider
When performing bright-field microscopy, pull the
slider out to the click position and bring the empty
position into the optical path. When performing
diascopic fluorescence microscopy, push the slider in
all the way and bring the BA filter into the optical path.
(3) Diascopic fluorescence objective
When performing diascopic fluorescence microscopy,
you can use three types of objectives: CFI E Plan 4x,
CFI E Plan 10x, and CFI E Plan 40x.
(4) Shielding plate
Attach the shielding plate to prevent strong light
including UV light from the specimen through the
condenser from flashing into your eyes.
2 Focus on the target using the bright field
microscopy method.
Follow steps 1 to 12 of the procedure in "2.1
Bright-field Microscopy", and focus on the target
using the 10x objective with the bright-field
microscopy method.
Chapter 2 Microscopy Techniques
72
BA filter slider
Shielding plate
Diascopic fluorescence
objective
• CFI E Plan 4x
• CFI E Plan 10x
• CFI E Plan 40x
EX filter slider