Using a compound light microscope
Appendix 8
Page 1 of 2
Always carry your microscope with two hands, one under the base and the other supporting the
top. Store your microscope in a safe (to prevent accident), covered (to reduce dust contamination)
place. If you get a damaged microscope, tell us immediately so that we can have it repaired.
The microscopes in the Biol 342 lab are similar to this, but not every attachment is shown in each
illustration. However, all compound microscopes have common features and require similar
set-up. None of the condenser parts or controls is shown in the photograph. Key numbers for
diagram: 1) objective, 2) slide in slide holder, 3) condenser aperture control, 4)!condenser
centering screws, 5) field diaphragm, 6) accessory lens for condenser, 7 and 8) controls for
transmitted light, 9) condenser focus.
Objective lenses vary in three major ways – the
magnification, the numerical aperture, and whether they
require immersion fluid. The numerical aperture is a
measure of the light-gathering characteristics of the lens,
which correlates with image quality, and generally is
higher for higher magnification objectives. The highest
numerical aperture generally available is N.A. 1.4.
High N.A. lenses often require immersion oil to collect all
of the light that comes from the specimen. If so, the
objective will have a black ring, typical for 100x objectives. Once you have focussed on your
specimen at 40x, swing the objective to one side (so that the specimen is between the 40x and
100x objectives) and then put a small drop of immersion oil on the coverslip. Swing the 100x
objective into place. You will only be able to use the fine focus control with this objective. Once
you have finished with this objective/slide combination, wipe them clean them with lens tissue and
cleaning fluid (provided). Do not get immersion oil on the 40x objective.
For slides sealed with nailpolish: When removing immersion oil from the coverslip after
examinations, do so very gently, lest your rubbing dislodge the coverslip or introduce leaks in the
nail polish seal. Saliva often works better for this than cleaning fluid or solvent.
Using a compound light microscope
Appendix 8
Page 2 of 2
Transmitted light microscopy
• Mount your specimen on a microscope slide and cover with a #1-thickness cover glass. For
relatively permanent slides, or for oil immersion, seal the edges with nail polish.Two thin
layers is better than a single thick one.
• Place the slide cover glass side up on the stage, and secure (#2 on diagram).
• Turn on the transmitted light using the knob (#8 on diagram). Make sure that there is light
coming through the field diaphragm (#5 on diagram).
• Adjust the distance between the oculars so that you can see through both of them. Like your
shoe size, this will be a constant value. Most microscopes have a scale that you can preset once
you know this distance. Adjust the focus on one of the oculars (knurled ring) so that you can
see clearly through both eyes. It is important to be as comfortable as possible to avoid eye
strain. You should sit with your back as straight as possible to avoid back strain.
• Using a low power (10x) objective, focus on the edge of the coverslip or nail polish seal,
which will be at a slightly higher level than your specimens. For small or colourless specimens
it can be difficult to find the proper focal level. This strategy gets you close to the correct level
very quickly. Continue to focus down using the fine focus.
• Check the condenser focus and centering. The condenser is a series of lenses and controls
below the stage (#3-5 on diagram). To do this, close the field diaphragm by rotating the collar
(#5 on diagram), and then look through the oculars at your image.
• The condenser can be out of adjustment in two ways: focus and centering. Adjust the focus
using the knob shown in #6 on diagram, so that the diaphragm edges are as crisp as possible
(see B, below). Centre the condenser using the two knobs (#4 on diagram) so that the image
resembles C, below. Then, open the field diaphragm until the edges are no longer visible (D,
below). This is called Köhler illumination, which is optimum for bright field transmitted light
•
microscopy. The condenser focus must be checked each time you change objectives.
The objectives on your microscope are parfocal, meaning that you can switch from one to
another without doing major refocusing. However, the 100x objective is an “oil immersion”
lens, meaning that you have to put a drop of a immersion oil (provided) between the coverslip
and objective for proper performance (the oil assists in the objective gathering more light). Oil
immersion lenses have a black ring on them; dry and water immersion lenses do not. After
using oil, both the objective and slide must be cleaned. The objective is cleaned with lens
cleaner and lens paper (provided). The other objectives are not oil immersion, and must be kept
clean and dry. When necessary, they are cleaned the same way as an oil immersion lens.