Microscope Terminology - Nikon ECLIPSE Si Instructions Manual

3.12

Microscope Terminology

(1) Total Magnification
The total magnification of a microscope is the individual magnifying power of the objective multiplied by
that of the eyepiece.
(2) Numerical Aperture (N.A.)
The numerical aperture is an important factor in determining the efficiency of the condenser and objective.
It is represented by the formula:
N.A. = n sin α
where n is the refractive index of the medium (air, immersion oil, etc.) between the objective and the
specimen or condenser, and α is the maximum angle at which light enters or leaves the lens from or to a
focused object point on the optical axis.
The larger the numerical aperture the brighter the image and the higher the resolution.
(3) Resolving power
The ability of an optical system to discriminate between two discrete objects separated by a minute
distance. The more minute the distance, the higher the resolving power of the optical system. In relation
to the numerical aperture, the resolving power is represented by the following formula:
Resolving power =
(The resolving power in the above table is indicated for λ= 0.55 μm.)
(4) Working Distance (W.D.)
The clearance between the front of the objective and the upper surface of the coverglass, when a
specimen image is sharply focused. Generally, the higher the magnifying power of the objective, the
shorter the working distance.
(5) Field Number of the Eyepiece
The diameter in mm of the viewfield observable through the eyepiece. When an eyepiece has an
indication of "10x / 20", it means that the magnification is 10x and the field number is 20 for that eyepiece.
(6) Real Viewfield
The diameter of the region of the specimen that is actually observed with the microscope.
Real viewfield = field number of eyepiece / magnification of objective
(7) Depth of Focus
The depth (thickness) of the specimen image in focus, extending above and below the focused image
plane. The larger the N.A. of the objective, the shallower the depth of focus.
DOF
= n ×
DOF
: Depth of focus (object side)
n
: Refractive index (according to the medium between the specimen and objective)
If the medium is air: 1
If the medium is immersion oil (Nikon Immersion Oil): 1.518
ω
: Resolving power of the eye (assumed to be 5' = 0.0014)
M
: Total magnification
N.A.
: Numerical aperture of objective
λ
: Wavelength (for visual observation, assumed to be 0.55 μm)
(8) Excitation filter
Of the light illuminating the specimen, an excitation filter allows only the wavelengths of light necessary to
emit fluorescence from the specimen to pass through. The term "excitation filter" refers to the excitation of
fluorescence in this process.
(9) Barrier filter
A barrier filter allows only the wavelengths of light consisting of excited fluorescence to pass through. The
term "barrier filter" is used because the filter acts as a barrier absorbing non-fluorescent light.
Chapter 3 Detailed Explanation
λ
λ: Wavelength
× Ν Α
2 . .
ω
λ ×
250000
+
N.A. M N.A.
× ×
2 2
97