Updated: May 5, 2015
AxioPlan Fluorescence Microscope
- Microscope: Axioplan with 2.5X, 10X, 20X, 40X and
60X oil objective lens
- DIC and phase contrast
- 100W HBO lamp with Fluor control. Common UV filtering cubes
- Digital CCD camera: AxioCam105 (Color, 5.0MP)
- Imaging software: ZEN 2.
- Conventional fluorescence microscopy for GFP-tagged or dye-stained cells or tissues. High quality
image-acquiring and -editing system.
microscope is usally a compound microscope equipped with epifluorescent
light path system, such as the Zeiss Axioplan in the following
computer-assisted digital imaging operations greatly facilitate the
users for acquiring images both in transmitted or fluorescent light
Transmitted light (bright field, BF) microscopy
your specimen on a microscope slide and cover with a
coverslip. For relatively permanent slides, or for oil
immersion, seal the
edges with nail polish.
- Place the slide on the stage, and secure.
- Turn on the transmitted light using the knob.
- Note that the
switch behind the knob should always be left with the top side pushed
is the main switch for the power supply to that light source.
- Make sure that there is light coming through the field
diaphragm. Sometime this is
left covered with a black cup, since transmitted light interferes with
microscopy (discussed later).
- Adjust the distance between the ocular lenses so that you
can see through both of them.
- Like your shoe size, this will be a
constant value. Most research microscopes have a scale that you can
- Adjust the focus.
- Using a low power objective (20x) focus on the edge of
– this will be at a slightly higher level than your specimens.
- Some specimens are small and colorless, so it can be
difficult to find the proper focal level. This strategy gets you close
correct level very quickly. Continue to focus down using the fine focus.
- Objective lenses vary in two major ways: the magnification
and the numerical aperture.
- The numerical aperture (N.A.) is a
measure of the light-gathering
characteristics of the lens.
- The highest N.A.currently available is 1.4 (in the 63x
lens); other objectives have lower N.A.
- The objective must be matched
to a lens in the condenser, selected by rotating the ring under the
- Sometimes you need differential
interference microscopy (DIC, discussed later) to increase the contrast
- The condenser
is a series of lenses below the stage (see the Figure at right).
- Close the field diaphragm by
rotating the collar (#5).
- Look through the oculars at your
- The condenser can be
out of adjustment in two ways: focus and centering.
- Adjust the focus using the condenser
(field diaphragm) focus knob (#6), so that the
diaphragm edges are as crisp as possible, as shown in the figure below (B).
- Centre the
condenser using the two knobs (#4) so that the image resembles (C) in the figure below.
- Open the field diaphragm
(#5) until the edges are no longer visible (as in D).
- This is called Köhler illumination,
which is optimum for bright field transmitted light microscopy.
- The condenser focus should be checked each time you change
- The objectives on microscopes are parfocal: parfocal means
that you can switch from one to another
without major refocusing.
- Note that the 63x and 100x objectives are “oil immersion”
- Oil immersion lenses require special immersion oil
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
- After using oil, both the objective and slide must be cleaned carefully.
- Use special lens cleaner and lens paper.
- Non-oil immersion lenses must be kept clean and dry.
- When necessary, they are cleaned the same way as an oil
- Some objective lenses use water, or liquid growth medium,
rather than oil, for immersion.
- Water immersion lenses have a blue ring.
- Growth medium should be rinsed off with distilled water,
then the objective should be blotted dry.
Differential interference contrast (DIC) microscopy
DIC microscopy uses polarized
transmitted light to increase the
contrast of images of unstained cells. This was developed by Nomarski,
also called Nomarski contrast. Briefly, the specimen is illuminated
polarized light, and after passing through the specimen and the
light passes through a second polarizing filter that is at an angle to
first. The polarity of the light is changed as it passes through the
leading to increased contrast and the appearance of a three-dimensional
There are four parts to the optical system:
- The condenser lens must be appropriate to the objective –
matched for numerical aperture N.A.
- The polarizer filter must be swung into the light path –
this is below the condenser, but above the field diaphragm.
- The analyzer filter (see “slider for DIC”; just above the
for epifluorescence) must be pushed into the light path.
- Sometimes, an additional small polarizing filter just above
the objective must be adjusted for maximum contrast, although this may
incorporated into the analyzer filter. Either way, as this ‘Wollaston”
is rotated the image becomes more or less contrasted.
substances (e.g. proteins or stains) absorb
light of one colour (wavelength), and then release that energy as light
different color (longer wavelength). e.g.
- fluorescent substances that absorb blue light typically
fluoresce (or emit) green light.
- fluorescent substances that absorb green light typically
- The Zeiss Axioplan Epifluorescent microscope is
equipped with a mercury vapor lamp light
source HBO-100, as shown in figure at right (A).
- The light from the mercury vapour lamp goes through an
aperture controlled by the slider (B) and one of a series of filter sets (excitation/emission
cubes, or dichroic mirrors, E ). The filter
sets are (left to right)
- I. G365/FT395/LP420.
- II. 450-490/FT510/LP520.
- III. BP546/FT580/LP590.
- IV. Empty.
microscopy, both the mercury
light Power Box and bulb lifespan-monitering control panel FloArc must be turned on.
- Once turned on, the
bulb should be left on for at least 20 minutes, so that it
completely warmed up.
- Once turned
off, it should be left
off for at least an hour until it is completely cool again.
- Mercury vapor bulbs are expensive
and fragile, and this heating/cooling regime improves bulb performance
- Find your specimen using transmitted light, and then cover
the transmitted light at the field diaphragm, or turn the bulb off.
- Pull out the DIC slider: this absorbs a lot of light.
- Choose the correct filter set (E) for your fluorescent stain or protein.
- Pull back the slider covering to admit ultraviolet light (C).
- Note: Generally, it is easier to use
epifluorescence in a
the digital camera-- for detailed information,
please see ZEN lite guide.
- IMPORTANT: Turn OFF the Power Supply and unplug
it from behind BEFORE changing the mercury bulb!
the Mercury bulb is used for more than its normal life span:
- the glass will turn black,
- the intensity may decrease
or vary (i.e.
- the intensity may be
greater on one side of
the field of view than the other,
- the bulb MAY EXPLODE!,
and cause mercury contamination!
- For more information on Mercury Bulb and Safty Regulations, please refer to Zeiss website.