ZEISS AXIOVISION
Software’s User Manual
Managed by
For information about this instrument, please contact
Dr. Alloysius Budi Utama @ ext. 8232 or e-mail budiutama@rice.edu
Use of AxioVison AC Software with Zeiss Axioskop Microscope
START-UP
When doing a start-up on this particular computer, it
might mention / ask about password changes -- ignore
this; there are no "real" passwords to use, so just click
though the queries and get past that (if they appear).
The icon for the AxioVision AC Rel 4.5 program is
usually in the center of the desk-top. When the
computer is started, the color AxioCam MRc5 camera is
activated (the small green light on the top of the
camera should be ON). If camera is not ON, check the
camera wire connection to the back of the computer.
Turn ON whatever microscope lamps (halogen and/or
mercury arc) are to be used. [A separate document, Use of the Axioskop, contains brief details
and descriptions of Axioskop microscope components and operation.]
PRE-IMAGE ACQUISITION
When the major Axiovision AC "working window" appears, click on the Live icon (in the "icon
strip" under Measure in main menu — red arrow). This will give the Live — AxioCamMRc5 "live
image box" that will have the image (red circle). You can also click the Workarea box (green
arrow) to show the workarea window (green circle).
Remember to pull OUT both silver metal "sticks" (below the
camera, on right side) for light to the camera from the objective
lens; push bottom stick IN for viewing through microscope ocular
/ eyepiece.
At the bottom left of the Live — AxioCamMRc5 box (circled in
blue) is the Objective box for whatever objective lens is being
used (for the correct micrometer scaling; e.g.. shown below is for
10x objective).
To view the overall image, View in the main menu has Zoom tool
options, and one can start with the Auto Zoom feature (shown
below). Auto Zoom has the advantage of easily displaying all of
the field of view within a reasonable-sized Live image window
box. The (+) Zoom and (-) Zoom features allow one to left click on
the image to quickly increase or decrease the image size. Right
click on image also has menu containing zooms etc.
Selecting Ruler will show the micrometer scale on the image frame at the x (upper) and y (left)
axis (see pg. 8).
For camera speed, "Slow" is preferred for stationary, fixed samples; the slower speed gives
better quality / higher resolution
images.
To get the working AxioCamMRc5 box (to the right of the large Live — AxioCamMRc5 image
box), click on the Properties icon (right-most icon at the bottom of the Live — AxioCamMRc5
image box — highlighted in light blue); when Adjust tab is selected from the menu, the
exposure time, white balance, and image histogram (pixel intensity / RGB scale) functions are
presented (see below, pg. 5). There are icons at the bottom of the Live — AxioCamMRc5 image
box for various scaling features related to brightness / contrast / image gamma (i.e., best fit,
min/max, linear, gamma 0.45).
Best Fit is: optimum of the minimum and maximum contained gray or color value excluding
specified minimum/maximum values.
Min/Max is: optimum of the minimum and maximum contained gray or color value.
Linear (Max. range) is: linear display of the full "dynamic" range of possible values.
Gamma 0.45 is: preset display curve for best color reproduction. Below, Linear is selected (light
blue highlight), but use whichever gives the truest or most desired image; Min/Max can be a
good start because it scales specifically over the particular pixel intensity range of the image.
The active image histogram display for each of these functions can be viewed by selecting
Display tab in the menu (to the left of Adjust tab). These scaling functions also can be selected
for by clicking in their respective boxes in Display. The use of features in the Display menu will
be mentioned in the post-image acquisition section, below.
Focus the sample's image on the screen, and choose a desired brightness level. [* note that, the
ocular lenses can be adjusted to give a focus for the user's eyes that matches the focus for the
digital image on the monitor.] remember, image brightness from the camera is determined by
both the lamp intensity setting (halogen) and the camera shutter exposure time.] The halogen
lamp, used for brightfield and DIC imaging, has an intensity adjustment knob which controls the
level of lamp brightness. A good starting point for halogen lamp intensity is usually between 5—
7; the exposure time can be chosen by sliding the bar horizontally with the left mouse click
depressed or entering a number with the keyboard (below, blue arrow). To make small changes
in time values, the left and right arrows can be clicked on. It should not be necessary to have
the lamp intensity at a value greater than 7; over 7 is too "hot." (Longer exposure time settings
tend to be a better way to increase image brightness.) When set at an average brightness or
"temperature" (approximately 4-6), the halogen lamp has a "black-body temperature" of about
3200K (as described below). If the 3200K box in the White Balance display area is used (below,
red arrow), the white balance will be adjusted to a basic setting based upon a light source
temperature of 3200K. This white balance adjustment is related to halogen lamp use (white
transmitted light), and affects the color balance of the sample. Note: There are neutral density
filters at the right base of the microscope; these will affect the color balance of the halogen
lamp. The true temperature of the lamp (3200K) is when these filters are not in place (sticks in
OUT position). If the mercury arc lamp is used (for fluorescence microscopy and imaging), white
balancing using a reference white background in the sample can not be directly performed
because there is no white light. [Light used for fluorophore excitation is of a particular
wavelength range (color); the sample's emitted light will also be of a particular wavelength
range (color).] As stated below, use of Interactive... or Automatic white balancing for
fluorescence can result in color distortion, and should be avoided. With the 3200K off, RGB
color balancing can be done manually using the RGB arrowheads or entering numbers in the
boxes (below, black arrow). If colors from the camera, as seen on the monitor, are still not the
actual colors seen in the microscope ocular / eye-piece, one can alternatively try the 3200K
standard setting (as mentioned below).
For brightfield / DIC imaging, do an interactive white balance by clicking on the "Interactive..."
box, and then use the cursor and left click mouse on a "background white region." The redgreen-blue (RGB) peaks in the image Histogram should ideally overlap (color balance), and also
be in the mid-region of the intensity grayscale (0 — 4095). As mentioned above, changes to the
image color can be done manually by clicking on the arrows next to the various color spots.
Under General in the menu of the AxiocamMRc5 box (below), the digital gain can be selected;
typically, an index value of 0 (gain factor of 1) is used (default). Image Orientation is Original
(default).
The background correction (Shading Correction) removes a digital background image which
may be noticed on the live image from the camera. [This kind of "background visual material" is
not due to material on your slide, but is in the camera / imaging system itself, and is a
"constant."] If you do not see or notice much "artifact" or annoying "material" in the
background, then you probably do not "need" to do a shading correction. However, if there is a
need or desire to do so: after doing the focus and brightness adjustments on your sample,
move to a region that does not have cells and is also very "clean and clear- (moving to a region
off the slide can work also). Click on the Shading Correction box - this will "clear or blank" the
background, and ideally nothing should then be seen in the image box. The Enable box should
then have a check. When you move back to the regions of your specimens (cells, tissue or
whatever), there should be