Darkfield Microscopy; Principles Of Oil Immersion Microscopy - Optika Italy B-510 Serie Manual De Instrucciones

12. Darkfield microscopy

B-510DK 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 12.1 and 12.2) and then we give a step-by-step
guide to the configuration of B-510DK (chapter 12.4).
General tips for immersion microscopy are also given.

12.1 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 re-
fractive 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 coverslip 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 vacu-
um and considerably lower than most liquids, including water (n =
1.33), glycerin (n = 1.470) and common microscope immersion oils
(average n = 1.515).
Practically, the maximum numerical aperture of a dry objective sys-
tem 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 where
individual light rays are traced through the specimen and either
pass into the objective or are refracted 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 coverslip. These rays are refracted at the coverslide-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 deviation due to refraction. The
numerical aperture is thus increased by the factor of n, the refractive index of oil.
Microscope objectives designed for use with immersion oil have a number of advantages over those that are used dry.
Immersion objectives are typically of higher correction (either fluorite or apochromatic) and can have working numerical
apertures up to 1.40 when used with immersion oil having the proper dispersion and viscosity. These objectives allow the
substage condenser diaphragm to be opened to a greater degree, thus extending the illumination of the specimen and
taking advantage of the increased numerical aperture.
A factor that is commonly overlooked when using oil immersion objectives of increased numerical aperture is limitations
placed on the system by the substage condenser.
In a situation where an oil objective of NA = 1.40 is being used to image a specimen with a substage condenser of smaller
numerical aperture (1.0 for example), the lower numerical aperture of the condenser overrides that of the objective and the
total NA of the system is limited to 1.0, the numerical aperture of the condenser.
Oil immersion and Numerical Aperture
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F ig. 21 F ig. 21