Darkfield Microscopy; Principles Of Oil Immersion Microscopy - Optika Italy B-380 Serie Manuel D'utilisation

11. Darkfield microscopy
B-383DK is a darkfield system specific for blood analysis with a 1.36 - 1.25 N.A. special extra efficient darkfield
condenser and a 100X plan-achromatic objective with adjustable iris diaphragm.
The X-LED illumination ensures the high level of light intensity typically needed in high magnification darkfield
techniques.
In order to correctly use this microscope, one has to gain some familiarity with:
a. oil immersion technique
b. darkfield technique.
In the following manual we present the basics of these methods (chapters 11.1 and 11.2) and then we give a
step-by-step guide to configuration of B-383DK (chapter 11.4).
General tips for immersion microscopy are also given.
11.1

Principles of oil immersion microscopy

The ability of a microscope objective to capture deviated light rays from a specimen is dependent upon both the
numerical aperture and the medium through which the light travels.
An objective's numerical aperture is directly proportional
to the refractive index of the imaging medium between
the coverslip and the front lens, and also to the sin of
one-half the angular aperture of the objective.
Because sin cannot be greater than 90 degrees, the
maximum possible numerical aperture is determined by
the refractive index of the immersion medium.
Most microscope objectives use air as the medium
through which light rays must pass between the cover-
slip protecting the sample and front lens of the objective.
Objectives of this type are referred to as dry objectives
because they are used without liquid imaging media.
Air has a refractive index of 1.0003, very close to that
of a vacuum and considerably lower than most liquids,
including water (n = 1.33), glycerin (n = 1.470) and com-
mon microscope immersion oils (average n = 1.515).
In practice, the maximum numerical aperture of a dry ob-
jective system is limited to 0.95, and greater values can
only be achieved using optics designed for immersion
media.
The principle of oil immersion is demonstrated in Fig. 21
Oil immersion and Numerical aperture
where individual light rays are traced through the speci-
men and either pass into the objective or are refracted
Fig. 21
in other directions. Fig. 21 (a) illustrates the case of a
dry objective with five rays (labeled 1 through 5) shown
passing through a sample that is covered with a cover-
slip. These rays are refracted at the coverslip-air interface and only the two rays closest to the optical axis (rays
1 and 2) of the microscope have the appropriate angle to enter the objective front lens. The third ray is refracted
at an angle of about 30 degrees to the coverslip and does not enter the objective. The last two rays (4 and 5)
are internally reflected back through the coverslip and, along with the third ray, contribute to internal reflections
of light at glass surfaces that tend degrade image resolution. When air is replaced by oil of the same refractive
index as glass, shown in Fig. 21(b), the light rays now pass straight through the glass-oil interface without de-
viation due to refraction. The numerical aperture is thus increased by the factor of n, the refractive index of oil.
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