Examination and processing of human semen - libdoc.who.int ...

APPENDIX 3 Microscopy 235
Note: If the microscope is trinocular (i.e. has a third ocular to which a camera can be attached for
photography or video-recording), there will be a light-deflection knob, which is generally located to
the right of the eyepieces. This knob is likely to have three settings: one to allow all the light to go to
the eyepieces, one to allow all light to go the camera, and a third that deflects half of the light to the
eyepieces and half to the camera.
Box A3.1 The objective lens
Each microscope lens has information on it, such as:
UPlanFl PlanApo Plan Neofluor Plan S Fluor
20×/0.80 imm corr 40×/0.75 Ph2 100×/1.35 oil iris 100×/1.25 oil Ph3 20×/0.75
160/0.17 /0.17 /– /0.17 WD 1.0
Explanations of the various markings are given below.
Plan: a planar lens, permitting a flat field of view, in which everything is in focus.
Apo: an apochromatic lens that is highly corrected for chromatic aberration.
F, Fl, FL, Neofluor, Fluo, Fluotar, UV, S-Fluor: a lens that will transmit UV light and is used for fluorescence
microscopy.
100×, ×63, 40×, etc.: the magnification of the lens.
0.30, 0.50, 0.80, 1.30, 1.40, etc.: the numerical aperture (NA) of the lens. This is an indication of the
light-gathering ability of the lens. Together with the wavelength of the light used (lambda), the NA
determines the resolution (the smallest distance between two objects that can be distinguished as
separate). NA = × sin , where (eta) is the refractive index of the immersion medium and (alpha)
is the angle between the edge of the cone of illumination and the vertical. As the maximum value of
sin is 1.00, the maximum NA is theoretically equal to , but in practice the maximum value is 1.4.
Choose the highest NA for best resolution.
Ph, Ph1, Ph2, Ph3, NP, N: indicates a lens with a phase ring in it. Ph indicates positive-phase rings
and NP or N negative-phase. Ph1, Ph2 and Ph3 lenses each require a different phase annulus in the
condenser. Positive-phase-contrast optics permit intracellular structures to be seen (used for wet
preparations and motility), while negative-phase-contrast optics produce white images against a dark
background (used for wet preparation vitality or CASA).
Imm, immersion, oil, W: indicates a lens designed to work with a fluid—often oil, water (W) or glycerol—between
the object and the lens to provide a sharper image. (If not indicated, the lens is “dry”
and should not be used with a liquid.)
Iris: indicates a lens with an iris controlled by a knurled ring.
Corr: indicates a lens with a knurled correction collar that allows the use of immersion media of different
refractive indices.
160, : the tube length or distance between the eyepiece and the objective. This is usually 160 mm
but in modern lenses can be infinity ().
0.17, –: the thickness of the coverslip required for the objective. Coverslip number 1.5 (thickness
0.16–0.19 mm) is useful for most purposes. Haemocytometers need coverslips number 4 (thickness
0.44 mm). “–” means that the thickness of the coverslip is not important or that immersion fluid can be
added directly to the slide.
WD: working distance; the distance from the front lens element of the objective to the closest surface
of the coverslip when the specimen is in sharp focus. The WD generally decreases as the magnification
and NA increase, giving rise to lenses with working distances that are normal (NWD, up to 5 mm),
long (LWD, 5.25–9.75 mm), extra-long (ELWD, 10–14 mm) and super-long (SLWD, 15–30 mm). Some
microscopes may require an LWD lens for use with an improved Neubauer chamber.
Refractive index: the extent of phase retardation of light as it passes through a medium. The refractive
index (RI, , eta) of a vacuum is 1.0000, of air is approximately 1.0 (1.0008), of water is 1.33, of
glycerol is 1.47 and of most immersion oils is 1.515. Mounting media after drying have similar refractive
indices (1.488–1.55) to that of glass (1.50–1.58).