Manual for the Axioplan Imaging (Brightfield)
For assistance contact Birgit Ehmer ([email protected]; 513-300-4801)
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Choose a day or a month in the
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Chick on add to choose a
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Sign up for confocal time II
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• Choose the start time and
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Microscope Parts
Becoming familiar with the different parts of the microscope and what they do will help
you to collect a good image quickly.
You will need the field aperture to set up Koehler illumination (see later).
The neutral density filters will decrease the brightness without affecting the color. The
number refers to the percent light passed.
The brightness knob will regulate the intensity of the illumination, but also affect the
color. If the brightness is set to very low the light will be yellow. The more you turn it up,
the closer is the approximation to white light.
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Microscope Parts
You need to focus the condenser only if you plan to acquire brightfield images, not
for fluorescent images.
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Manual Controls
•You can rotate the filter
cube turret with the two
buttons below the focus
knob on the left side of
the scope.
• The objective lens
turret can be rotated by
using the two buttons
below the focus knob
on the right side.
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Condenser settings
Choose condenser setting (DIC, Brightfield, Phase.
I/H for brightfield microscopy
2, 3 for phase contrast (there are no phase contrast
objectives available, so don’t use)
II, III for DIC (Differential Interference Contrast). Use II for
10x, 20x, use III for any higher magnification.
Moves the top condenser lens in or out.
The lens should be IN for all objectives except the 2.5
objective
Regulates contrast aperture. The default is maximum size
(use right button). While looking into the microscope you
can close the aperture to some degree if you find it
improves your image.
Generally the resolution is best with the aperture
completely open (max), the contrast is best with it closed
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(min).
Visualizing an unstained sample in brightfield
Brightfield. No contrast.
Brightfield. Contrast aperture closed.
DIC.
DIC. Different adjustment of prism.
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Koehler (or Köhler) Illumination
• Koehler illumination occurs when the condenser and
field iris are positioned to optimally illuminate the
specimen.
• Koehler illumination needs to be done for brightfield,
darkfield, phase contrast, and DIC.
• First step: Choose the appropriate objective and focus
on the specimen.
• If the next steps are done without the specimen being
focused, then Koehler illumination will not be
achieved.
• Koehler illumination must be rechecked if a different
objective is used.
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Koehler Illumination
• Adjust the condenser to
its highest position using
the condenser focus knob
on either side of the
microscope.
Condenser focus
Condenser
focus
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Koehler Illumination Close Field Iris
•
•
•
To adjust the field iris, use
the dial that is on the lower
right of the scope.
Close the field iris.
If the condenser is not too
badly aligned, you should
see the edges of the leaves
of the iris.
Open Iris
Closed Iris
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Focus the field iris
• Adjust the condenser so that
the field iris is in sharp focus
using the condenser focus
knob on either side of the
microscope.
Unfocused Iris
Focused Iris
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Koehler Illumination Center the Condenser
•
•
•
•
The field iris may be off center
when view through the ocular
lenses.
Centering is easier if you open
the iris so that its edge almost
reaches the edge of the field of
view.
Center the image of the iris
with the condenser centering
screws that are located on
either side, and just under, the
condenser (Only the left screw
is shown).
When you are done centering,
open the iris until it is just
outside the field of view.
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Microscope control via Software
Top condenser lens in/out. Use lens except for 2.5x objective
Choice of condenser. Use brightfield or DIC
Control contrast aperture. Contrast is best using low values, resolution is
best using high values. For stained samples use a value close to 1.
Select neutral density filters. Use to decrease light without affecting color.
Number refers to percent of light transmitted.
Control field aperture. You need this for Koehler illumination.
Switch halogen lamp on/off
Control brightness of halogen lamp. Use at least 3 volts, otherwise light
will be very yellow
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Start up
You don’t need to switch on the mercury light if
you are just using brightfield.
If you want to look at fluorescent samples, switch
on the mercury light (FluoArc)
If you switch a mercury light on it should stay on
for at least half an hour.
If you switch a mercury light off it should cool off
for at least half an hour before you switch in on
again.
Switch on the microscope (green button on right side)
Log into the computer:
Username:lab
Password:superpig
Start up program.
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Choosing Camera
There are 2 cameras connected to the
microscope.
MRM black&white camera. More light
sensitive, use for fluorescence
MRc5 color camera. Use for brightfield
To switch between cameras:
1) Select camera on the AxioVision Menu
2) Turn black knob on top of microscope:
Counter clockwise: black&white camera (MR)
clockwise: color camera (MRc5)
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Microscope Parts III
Pull the camera slider (arrow) all the way out before you take a picture to direct the
light to the cameras.
Turn the black knob (red circle) to select the camera you want to use:
counter clockwise: black & white camera. Use for fluorescence.
Clockwise: color camera. Use for brightfield images.
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Basic photography (brightfield)
It is fastest to control the microscope and the cameras using the workarea. If the
Workarea window does not show at startup you can select it
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Basic photography (brightfield)
Select “Live” to see the image from the camera
select “cameras-AxioCamMRc5-Adjust”
Click on “Measure” in the camera menu to select
an appropriate exposure time
You should see an image of your sample on the
screen.
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White balance (brightfield)
To get the correct color you need to do a White Balance. You should acquire a new white balance
at the beginning of each session and whenever you change the intensity of the illumination.
First move on your slide to an empty area on your slide.
Then choose “White balance” on the bottom menu bar of the live view window. Move back to
your sample after the white balance is done.
Before white balance
After white balance
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Scaling
In order to scale your
image you need to tell the
program which camera
and which objective you
are using. Please note
that the scaling is not
identical for the color and
the black and white
camera.
Choose the magnification
and the objective which
applies to your image and
either click on “activate”
or “apply to image”
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Basic photography (brightfield)
After you acquired a white balance you need to measure
the exposure time again.
You can adjust the brightness of the image by increasing
or decreasing the exposure time.
You will get best results if 80% is selected.
Focus your image by turning the focus knob on the
microscope while looking at the live view on the computer
monitor.
When you are happy with your image click on “Snap”
either in the live view menu bar or the top menu bar.
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The green bar at the bottom of the
live view window is a focus help.
Click on green bar to start.
Change focus. If the green bar
expands towards the right, the
focus is getting better.
If the focus is getting worse, the
red line will indicate the best focus
achieved until then.
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DIC
You must rotate the dial on the multipurpose condenser to “II” or
“III” in order to set the condenser for DIC with any of the
objectives that we have. III will let you do DIC with objectives
that have an NA of ~0.7 or higher. Selecting “III” places the
appropriate polarizer and a prism under the sample.
In addition you need to select the DIC filter cube. Select #2. Or
select “Analyzer Transm” as the filter cube under the “Reflected”
tab in the microscope menu.
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DIC – Prism Adjustment
•You can move the objective Nomarski Prism
orthogonal to the light axis using a screw on the
prism insert on top of the objective lens.
• Depending of the position of the prism the
dark and light areas can reverse, so convex
structures will look concave. In addition the
background intensity changes.
•Adjust the screw until you get the image you
want.
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Visualizing unstained sample in light microscopy
Brightfield
Phase Contrast
For stained objects
For thin, unstained objects
DIC
Darkfield
3-D effect for unstained
objects, thick and thin
For particulate matter like silver grains
Polarization
Andrew Davidhazy
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Difference between phase contrast and DIC
Generally DIC is preferable as it will give you better resolution, but DIC won’t
work if plastic material is in your light path e.g. cells grown on plastic tissue culture
dishes.
Phase
contrast
DIC
images and table from molecular
expressions website 27