Dark-Field Microscopy - Nikon ECLIPSE Si Instructions Manual

2.5

Dark-field Microscopy

Dark-field microscopy is a method of using oblique illumination to observe unstained specimens. A bright
observation target can be visualized on a dark background in high contrast.
In dark-field microscopy, the annular diaphragm narrows the illumination to an annular shape, and light
passes through the edge of the condenser lens to obliquely illuminate the specimen. Using an objective with a
smaller numerical aperture than the condenser lens allows only light scattered by the specimen to pass
through the objective to form a dark-field image.
For details on the configuration and the assembly of the microscope for dark-field microscopy, see "4.2.5
Configuration for Dark-field Microscopy."
1 Prepare the microscope.
Check that the following optical parts are usable.
(1) Slider for dark-field microscopy
When performing bright-field microscopy, bring the
empty position into the optical path, and when
performing dark-field microscopy, bring the dark-field
position into the optical path.
(2) Objective for dark-field microscopy
Check that the objective is attached to the nosepiece.
Combination of optical parts
There are limits on the NA values of objectives that
can be used with dark-field microscopy.
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.
3 Insert the dark-field slider and bring dark-field
position into the optical path.
Insert the dark-field slider from the right side up to the
click position, and bring the dark-field position into the
optical path.
The dark-field annular diaphragm is brought into the
optical path. The microscope is now ready for
dark-field microscopy.
Chapter 2 Microscopy Techniques
76
Objective for dark-field
microscopy
Slider for dark-field
microscopy
Bring the objective for dark-field microscopy
into the optical path.