Darkfield Microscopy; Principles Of Oil Immersion Microscopy - Optika Italy B-510 Série Manuel D'utilisation

11. 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 11.1 and 11.2) and then we give a
step-by-step guide to the configuration of B-510DK (chapter 11.3).
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 co-
verslip 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 maxi-
mum possible numerical aperture is determined by the re-
fractive 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
vacuum and considerably lower than most liquids, including
water (n = 1.33), glycerin (n = 1.470) and common microsco-
pe immersion oils (average n = 1.515).
Practically, the maximum numerical aperture of a dry objec-
tive 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. 20
where individual light rays are traced through the specimen
and either pass into the objective or are refracted in other
directions. Fig. 20(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 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. 20(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.
Oil Immersion and
Numerical Aperture
Page 14
Fig. 20