Download Microscopes:

Microscopes:
Care and Usage.
PJ Chenoweth
Kansas State University
Contents
Introduction
Basic Knowledge and Techniques
Types of Microscopes
Terminology
Types of Microscopes
Care of Microscopes
Alignment of Microscopes
Cleaning Microscopes
Introduction:
The microscope is the most important quality piece of equipment used for semen
evaluation. Large differences in results obtained for both sperm motility and morphology
estimations have been attributed to microscope quality. Microscope “quality”, in turn, is a
function of the optical quality of microscope components, their alignment and care.
While it is possible to over-economize in relation to the first factor, the contribution of
the last two factors to the accuracy of results is often under-estimated.
Basic Knowledge/Techniques :
Understanding basic light/microscope theory
Identify microscope parts and their functions
Cleaning the microscope
Centering the light source to obtain even illumination (diaphragms, condenser)
Types of Microscopes
A variety of optical systems, configurations, and manufacturers are in existence.
Practitioners could be excused if they despair over which is the best, most economic
choice for the task in hand. For effective semen evaluation, several generalizations may,
however, be made. The 3 types of microscopes routinely employed for semen evaluation
in livestock are brightfield, phase contrast and differential-interference phase. A list of
highly recommended features are as follows:
Binocular
100, 400 and 1000+ total magnification (objective x eyepiece).
Mechanical stage (preferably warmed)
Robust
Sealed, protective carry case
Terminology
Compound microscope – a microscope incorporating more than one lens to increase
magnification over that obtained with a simple (1 lens) microscope.
Achromatic – A term which, when appled to the lens, means that it brings in light from 2
different parts of the spectrum to the same focus, thus reducing chromatic abaerration.
Aperture – numerical representation of the resolving power of an objective.
Numerical aperture (NA) – a term representing the angle of light accepted by the
objective (lens). Generally, the highter the NA, the greater the resolving power..
Brigthfield illumination – bright white light which illuminates a transparent or translucent
specimen to contrast against a white background.
Darkfield illumination – where the specimen is seen as a bright object against a dark
background.
Condenser – provides an even cone of light that illuminates the specimen. Light from the
condenser converges at the plane of the specimen, passes through it, and then diverges to
form an inverted illuminated cone which is captured by the objective lens. The most
common condenser used is the Abbe. Here the condenser NA should be equal to, or
greater than, the highest objective NA (usually 1.25 to 1.32 for a 100x oil objective).
Diaphragm – usually a circular disc containing 5 or 6 apertures of different sizes.
Köhler Illumination – a technique for illuminating a field from non-uniform light source.
Field of View – the visible area visible through the eyepiece when the microscope is in
focus.
Depth of Field – the distance along the optical axis in which the object can be viewed
with clarity. Generally, depth of field increases with decreasing aperture and with
decreasing condenser distance from the sample.
Magnification – total magnification, as observed through the eyepiece, is a function of
both lens and eyepiece magnifying power. Thus, 1000x is usually 100x (lens) multiplied
by 10x (eyepiece).
TYPE OF MICROSCOPES:
Bright-field.
This is the conventional microscope which is most widely used in the field. It is of
particular value for examination of stained smears, especially when used at 1000 x total
magnification or greater (usually employing an oil immersion lens).
Phase –contrast.
Several different forms of phase-contrast are available.
Conventional phase is advantageous for looking at live semen, as well as semen “fixed”
in an appropriate liquid fixative (e.g. Formal-saline or PBS-Gluteraldehyde). The
additional contrast provided by phase means that differential staining is usually not
necessary. Phase can be used effectively at high magnification (400-1000 x), where it can
depict some abnormalities (eg diadem defect, knobbed acrosomes) than conventional
bright-field techniques, although it is particularly useful in determining motility at 100250 x. Dark-field phase (giving an image produced by scattered light) s also very useful
for this determination.
Differntial-Interference-Phase (DIC). This represents the current “gold standard” for the
effective depiction of a number of sperm abnormalities (usually at 1000 x plus). Cost and
complexity
Combination Microscopes.
A number of microscopes are now available which combine a number of the above
features.
Care of Microscopes
No matter the quality (and expense) of the optics employed, a microscope is only as good
as its care and maintenance will allow. It is highly recommended that microscopes be
cleaned in a regular and appropriate manner, and that they be routinely serviced by a
trained microscope technician.
Some No-Nos
Make sure that all using the microscope are trained in its proper usage.
Try to avoid high temperatures, direct sunlight, dust, humidity, vibration, rough handling
Do NOT use ordinary tissue or even kimwipes to clean objectives (lenses). Rather, use
lens paper that is specifically made for that purpose.
Do NOT use any oil except the correct microscope oil for oil-immersion lenses. Note that
a number of these oils are made for specialized tasks (such as fluorescent microscopy).
Avoid vibration as much as possible. Use of a firm base, as well as placing mouse pads
under the microscope “feet” are helpful. Here, heavier microscope stands have
advantages, even if they are less portable.
Do NOT interchange optics between different brands/types of microscope.
Do NOT use the aperture diaphragm to control light intensity through the eyepiece.
Alignment of Microscopes
Focusing:
Use dry objective.
Focus specimen.
Rack condenser up to its highest position.
Remove the eyepiece and move the aperture diaphragm lever (on the condenser) to
reduce the light cone to approximately ¾ of its full open diameter. Then adjust this
diameter until it equals that of the objective. When these are equal, this is called “full
cone” and it represents maximum resolution.
Reducing the aperture diameter from that of full cone will reduce resolution, but increase
contrast and depth of field. The optimal position of the aperture diaphragm is a balance
between resolution and contrast and depends on the contrast and absorption detail of the
specimen. The tendency is for unskilled operators to close the aperture diaphragm too far
and increase contrast at the expense of resolution.
IT IS IMPORTANT TO NOTE THAT THE APERTURE DIAPHRAGM SHOULD BE
RESET FOR EACH OBJECTIVE FOR OPTIMAL RESULTS.
Cleaning Microscopes:
Use a dedicated lens cleaner with lens tissues. The latter should be kept dust-free. Both
facial tissues and Kimwipes tend to be too rough for lens use.
Problem Areas:
Front surfaces of eyepieces – grease from eyelashes.
Front lens of objectives – dirt, fingerprints, oil.
Top lens of condenser – oil, dust, debris.
Glass dust cover over base light – dust, debris, grease.
Blow off dust with syringe, bulb, pressure pak.
Note – rubbing lenses with a cloth or tissue in a dry environment will cause static
electricity and adherence of more dust and debris.
Useful Websites:
http://www.az-microscope.on.ca/glossary ofterms.htm
http://www.olympusmicro.com/primer/anatomy