Zeiss AxioImager A1 Assembly instructions

Instructions for Zeiss AxioImager
A1 Microscope for Stereology
Basic operation instructions as of 10/1/2009
The Stereology Workstation
By Jon Ekman

Start-up
1. Turn MASTER switch on Belkin Surge-
Master. This will turn on the LUDL stage
controller, microscope stand and the
Olympus Microre color CCD camera.
2. Turn ON X-Cite 120 Hg lamp if needed.
Note: The X-Cite 120 must stay ON a
minimum of 20 minutes. It also requires
20 minutes to cool down. If you turn it
o, the internal logic chip will not let the
system arc until it is cooled down.
3. Log in to the computer.
4. Load the Stereo Investigator or Neu-
rolucida software (shortcut on desktop
after training).
Once the software is up:
Set reference point in Live image in
software. This will make the live window
and mouse curser more responsive to
movement.
Load a sample and nd
focus...
1. Load a sample: Rotate the 5x lens into
position, then press the stage speed
button on the joystick to move the stage
out. Load your sample. Move the sample
back under the objective. Push in the
push/pull rod to deect light to the
binocular tube. Use coarse focus on the
right side of the stand and/or the ne
focus control on the joystick controller.
See joystick image to right for help.
2. Select objective in the software so
the software knows what objective you
are using.
3. Select Imaging Method:
For conventional brighteld viewing:
• Rotate the Condenser Turret Disk to the
H position.
• Swing out the polarizing lter located
under the condenser and remove the
full wave lambda plate if present.
• Align the microscope for Koehler Il-
lumination.
For Transmitted dark eld contrast
microscopy:
Using the 10x, 5x or 2.5x objective, you
can set the Condenser Turret Disk to D
after doing the steps above including
Koehler Illumination.
For wideeld uorescence viewing:
• Press the RL Key.
• Set Reector Turret to (2) Eyes DUAL
470EX & 560EX/515EM & 620EM
• Adjust the Incident Iris Stop Slider until
light intensity is adequate.
• Find Focus preferably on a dye that
emits in the green range (500-530nm).
• Pull out the Push/Pull rod to deect
light to the camera.
Start-up . . . . . . . . . . . . .
1 . . . . . . . . . Turn ON Master Switch
2. . . . . . . . . . Log on to Computer
3. . . . . . . . . . . . . Load Software
Optional . . . . Turn ON X-Cite Hg Lamp
Align Optical Components . . . .
1. . . . . . . . . . Koehler Illumination
Calibrate System . . . . . . . . .
1. . . . . . . . . . . . . Grid Tune Lens
2. . . . . Parcentric Parfocal Calibration
Run Experiment . . . . . . . . .
1. . . . . . . . . . . . . Set up Subject
2. . . . . . . . . .Set scope to Low Mag
3. . . . . . . .Trace Region(s) of interest
4. . . . . . . . . Set scope to High Mag
5. . . . . . . Dene Counting Frame Size
6. . . . . . . . . Dene SRS Grid Layout
7. . . . . . . . .Dene Disector Options
8. . . . . . . . Save Sampling Paramters
9. . . . . . .Select Markers for Counting
10 . . . . . . . . . . . . Count Objects
11 . . . . . . . . View Sampling Results
Fluorescent Imaging
Turn ON X-Cite 120 UV lamp
Note: For Fluorescence imaging:
Check the Reector turret is set to desired
excitation/emission.
• Reector Turret set to:
(1) Transmitted Light/Transmitted Pol.
(2) Eyes DUAL 470EX & 560EX/515EM & 620EM
(3) Triple Camera only Triple EX & EM
(4) Quad Camera only single BP
(5) Blank
(6) Reected light
• Set Reected light shutter to ON
and transmitted light shutter to OFF
Transmitted Light Imaging
The green LED located on the lower right
side of the microscope base indicates both
microscope power and that the transmitted
light shutter is ON. The light intensity control
knob will attenuate halogen power.
Set the Reector Turret to position (1) Trans-
mitted Light/Transmitted Polarized light
Set the condenser to H for Bright-eld viewing
or D for Dark-eld. Remember, depending on
the objective chosen, you may have to swing
out or swing in the condenser front lens. 2.5x
and 5x the objective front lens should be
swung out.
For polarized light imaging a full wave com-
pensator is available.

Align Optical Components
Koehler Illumination
PROCEDURE: Transmitted Light
1. Focus on specimen with the objective
to be used for data collection.
2. Close down the luminous eld dia-
phragm (1) while viewing.
3. Raise or Lower condenser vertical
control slightly (2) until luminous eld
diaphragm image is in focus.
4. Center luminous eld diaphragm im-
age using condenser centering screws
(3).
5. Open luminous eld diaphragm (1) to
edge of eld.
6. Adjust contrast using condenser
aperture diaphragm slider (4). In most
cases adjust aperture diaphragm slider
to mark matching the NA of the objec-
tive selected. For 20x, 40x, 63x and 100x
use step 7.
7. Remove eyepiece (5) and check to see
that 75%-80% of visible aperture is lled
with light (more light=better resolution
but less contrast).
Note: this should be done every time an
objective is changed.
Benets of Aligning Optics:
• Evenly illuminated image.
• Brilliant image without reection or
glare.
• Minimum heating of specimen.

Calibration
The system is calibrated by ITG sta, and
new users get these initial calibration
settings at rst login and startup.
The calibration is done using transmit-
ted brighteld method.
Calibrate only the objectives you plan to
use in your experiment.
Recalibrate only if you notice drastic
errors in software contour placement
between objectives.
Oil is not needed to Grid tune lenses.
Setup
1. Log in to the computer.
2. Load the Stereo Investigator or Neu-
rolucida software. (Shortcut on desktop
after training)
3. Load the calibration grid slide. Select
the 10x objective on the microscope
and in the software.
4. Push in the push/pull rod to deect
light to the binocular tube.
5. Move the small 25mm grid into center
view and focus.
6. Mouse click on the right top corner
in the software image window to mark
a the reference point. This will help nd
the small grid when you are ready to
calibrate higher power objectives; just
select move to reference point and the
software will drive stage to the location
of the small grid. Another option is to
count 8 squares in from top or bottom
of large 250mm grid, and then move left
or right until small grid is found.
7. Move to the large 250mm grid and se-
lect the lowest power objective you plan
to use in both the microscope and in the
software. Align for Koehler Illumination.
Grid Tune Calibration
8. Select Tools > Grid Tune Current Lens
9. Tell the software the box size:
Large grid =250mm
Small grid =25mm
10. Drop the anchor at intersection so
vertical dashed line is to the right of the
vertical grid line. The horizontal dashed
line should be just above the horizontal
grid line. The horizontal and vertical
Parcentric Parfocal Calibration
Move to reference point or nd the up-
per right corner of small grid.
Do not move the stage during parcentric
parfocal calibration.
Start with highest power objective focus
on corner of small grid.
1. Select Tools > Parcentric Parfocal
Calibration.
dashed lines should lay on the edge of
highest contrast of the actual grid im-
age.
Check the layout of the dashed grid
lines along one vertical column and one
horizontal row; ensure they mirror the
placement of the dashed lines passing
through the origin or anchor point. Ad-
just dashed lines if necessary. Right click
and select nished gridtuning lens.
11. Repeat on all other lenses to be
calibrated. Note the default cong does
not have the 100x oil or 50x oil on the
microscope though they have basic
calibrations set in the software.
2. Discard all unnecessary lenses. NL
and SI both use video lenses. Select OK.
3. Follow software prompts. Do not
touch stage X-Y controls or move joystick
X-Y during Parcentric Parfocal steps.
Once completed a dialogue will appear,
ensure all boxes are checked (default).

Stereology with the Optical Fractionator Workow
Tissue Preparation
The optical fractionator requires fairly
thick tissue. Thicker tissue allows one to
distinguish between cells that are on top
of each other while focusing through
the tissue making it easier to see the
cells clearly as they start to come into
focus. Optimal thickness for this method
is 20 microns after accounting for shrink-
age from tissue processing.
Options here are:
1. Cut thicker sections.
2. Dry the tissue as little as possible.
3. Avoid unnecessary alcohol steps.
Collect serial tissue sections without
losing any sections along the way. The
Optical Fractionator requires a series of
sections, such as every 3rd section. The
interval will depend on the size of the
structure and how many cells the struc-
ture contains; this can be determined
with a pilot study.
Do a Test Run or Pilot Study
A pilot study will help decipher the best
parameters to use for each experiment
including counting frame size, grid size,
and serial section interval. Due to vari-
ability across animals, a pilot study may
be required on one animal from each
group within the experiment.
With the Optical Fractionator the target
is to count no less than 200 cells per
animal. This can be achieved by count-
ing 10 sections with 10 sampling sites
per section and on average 2 cells per
sampling site. Make sure the Coecient
of Error (CE) is below 0.1. The best way
to approach a pilot study is to err on
the side of over-counting. Once cells
counts have been completed and the
CE has been calculated for the test
animal, you can decide whether or not
to adjust the parameters.
Once the results of a pilot study, have
been reviewed, adjustments to the pa-
rameters can be made in order to count
more cells or fewer cells as necessary.
Ways to DECREASE the number of
cells:
1. Make the counting frame smaller.
2. Decrease the grid size creating fewer
sampling sites.
3. Increase the section interval, for
example, counting every 4th section
instead of every 3rd.
Ways to INCREASE the number of
cells you count:
1. Make the counting frame bigger.
2. Make the grid size smaller so that
you visit more sites.
3. Decrease the section interval, for
example, counting every 3rd section
instead of every 4th.
Once good parameters for an experi-
ment have been determined, they can
be saved and quickly pulled up in the
workow for subsequent animals. Do-
ing a pilot study will ensure that quality
data is generated by obtaining the best
estimates of total cell number.
Step 1: Set up the subject
1. Number of sections to count: De-
fault is 1; new sections can be added as
you go.
2. Section’s cut thickness: How thick the
tissue was cut at.
3. Section Evaluation Interval: This is
the interval between the sections to
count. Every other section =2, every 3rd
=3 ...
4. Starting Section Number: Default;
start counting at section 1. The rst
section is dened as the rst section that
displays the desired morphology.
5. Z-value of First Section: This will
always be 0.00 at section 1. If starting on
a section other than one, it will be an in-
terval of the cut thickness. For example
if tissue was cut at 50 microns and you
are starting on section one, the Z value
=0.00, if starting on section 2 the Z value
=50, if starting on section 3, the Z value
= 100, and so on.
Step 2: Set microscope to Low Magni-
cation
Choose a low power objective (2.5x, 5x
or 10x) best tted for tracing a contour
around your region of interest (ROI).
Step 3: Trace your Region of Interest
(ROI)
Select a contour to use and then trace.
Use this same contour choice for the
same region of interest in subsequent
sections. More than one contour can
be traced to count dierent regions in a
structure or dierent structures. Con-
tours can be named uniquely.
Hint: Name contours after your area of
interest so they can quickly be selected
in the Display menu. Click on Display Set-
tings and then the contour tab. Click on
the contour to rename. The change will
be reected in the workow.
Step 3 is a good place to edit a contour.
Go to the Edit menu and click on Select
Objects. Then click on the contour and
it will be highlighted with white squares.
Once it is highlighted, right click for
various editing tools. Remember to right
click and Exit the Selection Tool once
editing is complete.
Step 4: Set Microscope to High Magni-
cation
Select a high power objective that will
be good for counting (40x-oil, 63x-oil or
These Parameters should be set so
that you end up counting at least
200 cells and/or a CE of less than 0.1
is achieved without spending time
counting more cells than you need to.
Also, do not change parameters for
every section within an animal! All
parameters must be kept constant
throughout all the sections of the
animal for the calculations to be
valid. It is best to keep the same pa-
rameters for each group of animals
as well.
NOTE: counting frame size, grid size, and serial section interval
Information below is derived from MBF Bioscience correspondence

Optical Fractionator Workow Continued...
100x-oil). Objective choice will depend
on tissue type and staining. Note: objec-
tives with magnication lower than 40x
will not give you enough depth of view
to count accurately especially when cells
are close to each other in Z.
Step 5: Measure Section Thickness
The calculations for Optical Fractionator
require the measured thickness of the
tissue post processing. Ideally, measure
the tissue thickness at every sampling
site for the most accurate cell estimates.
Measured thickness from all counting
sites will then be averaged and used in
the calculations.
1. Check the Measure the section while
counting option and leave the other op-
tions unchecked.
2. Set your Evaluation Interval for
measuring to 1. Every site will require a
thickness measurement.
Step 6: Dene the Counting Frame
Size
Set counting frame size to have 1-5
countable objects within.
Counting Frame tip: Choose a unique
identifying point of your cell or object
of interest, such as cell top, nucleus
top, nucleolus, widest cell perimeter, or
widest nucleus perimeter. Consistently
count objects only when the identifying
point is in focus.
Remember, also that the counting frame
can be placed during this step and the
software will always display it where you
placed it. Click and drag in the center,
or anywhere on the screen and that will
be the location (click) and size (drag)
that will be used while using the optical
fractionator.
Step 7: Dene SRS Grid Layout
Set the grid size so that there are ap-
proximately 10 counting frames in the
region of interest (ROI). Remember, this
is just an approximation. The number
of counting frames per ROI will never
be constant, because contours change
size in each section and because the SRS
Grid Layout is randomly thrown down
before every run to help reduce Bias.
If the sections have a history of hav-
ing very uniform thickness, choose to
set the Evaluation Interval to be larger
than one. Possible options could be
at every 3rd sampling site. This can
be done to save time.
As for Guard Zones, it is recommend-
ed that the guard zone be a minimum
of half the thickness of the object
being counted.
NOTE: Section Thickness measurements & Guard Zones
Step 8: Dene Dissector Options
Set Guard zones so that an over- or
under-estimation of cell totals does not
occur. Do not count cells that fall into
the guard zone. Guard zones are used
to ensure objects counted are not from
an area where potential knife damage
can occur either by being cut in half, or
plucked from the tissue during micro-
tomy. In practice, we recommend that
the guard zone be a minimum of half the
thickness of the object you re count-
ing. The dissector height will dene
the depth in which you will count cells.
This will also depend on how thick the
tissue is. For example, a section that is
20 microns thick after processing, could
have a top guard zone of 4 microns, with
a dissector height of 12 microns, leav-
ing by default a bottom guard zone of 4
microns. If tissue varies a lot in thickness,
choose a dissector and guard zones that
will t into the thinnest piece of tissue.
Step 9: Save Sampling Parameters
Save the sampling parameters that were
determined in the pilot study. Then for
subsequent animals, choose the saved
sampling parameters from Step 1: Set up
the Subject and skip steps 5-9.
Step 10: Select Markers for Counting
Choose a marker for counting and use
this same marker for subsequent sec-
tions. Change the name of the marker
to match the type of cells counted by
going to the Display menu, Display Set-
tings, and the Marker Tab. Click on the
marker and rename it. This name will
then appear in the marker tool bar and
will be easily visible for you to select it
for counting. On reload of a Data set the
software will ask to use marker names if
they are not present.
Step 11: Count Objects
In this step choose from a list of the
sections and contours that were drawn
for each section, by highlighting the con-
tour of a specic section to count and
click on the blue arrow. The stage will
move to the rst site and the software
will prompt to measure the top and the
bottom of the tissue. To nd the top of
the tissue focus up past the top of the
tissue until it is completely out of focus.
Slowly focus back down onto the tissue
and as soon as something on the tissue,
whether it is a cell or connective tissue
comes into focus. This is the top of the
section. To measure the bottom of the
section, focus all the way down through

DO NOT save each section as a
new le. Use the workow to add
new sections, as described above,
until all the sections in an animal
are traced and counted.
NOTE: One animal = one le.
and past the bottom of the tissue until
it is completely out of focus and then
slowly focus back up until something
just comes into focus. This is the bottom
of the tissue.
Note: If more than one person is doing
the counting for one experiment, all the
counters should sit down and come to
a consensus as to what is the top and
bottom of the tissue because if they
choose dierently, the dierence will be
reected in the results.
As far as choosing cells to count (place
a marker on), pick a point of the cell
(such as the top of the cell, the widest
perimeter of the cell, the nucleus, the
widest perimeter of the nucleus or the
nucleolus) and when that point of the
cell comes into focus, place a marker on
it, as long as it falls into the rules of the
counting frame discussed in step 6 and
falls within the dissector height (shown
as green in the Z meter). If this point of a
cell comes into focus while in the guard
zone (shown as red in the Z meter) the
software will block counting it.
Once all the sites have been visited, click
on Add New Section in the workow
below the blue start counting arrow.
The software will then return you to step
2 in the workow for the next section.
Follow the workow down to Step 11
again. Continue counting each sec-
tion and adding new sections until all
the sections the particular animal are
completed.
Step 12: View Sampling Results
Once counting is complete, the best way
to view the results is to click on Display
Probe Run List. Once this list pops up,
highlight all the like contours in each
section. For example, if there is only one
contour per section, highlight all of them
(hold down the Ctrl key to select more
than one) and click on View Results. If
the sections have mirrored contours
Left and Right, highlight all the left con-
tours and View Results. Later, highlight
all the right contours and View Results
for that. To see the results for the com-
bined contours, highlight all the right
and left contours and View Results.
Interpretation of Results
The rst items to appear in the results
window are the parameters for your
study. Marker will show the estimated
total cell numbers. Do not be alarmed
if some of the results are 0. Here is the
explanation of the dierent results:
Estimated total by optical fractionator:
If you manually entered the thickness
at the beginning of counting (and did
not measure while counting), then you
use the rst output in display probe run
list. This calculates the cell number and
bases the counts on the manual thick-
ness that you entered prior to doing the
cell counts. This is the *LEAST accurate
estimated value* unless you are 100%
sure of the thickness of all of your sec-
tions. If you did not enter the thickness
manually this number will equal zero in
the results, which can be changed by
clicking on Edit Mounted Thickness.
Estimated total mean measured thick-
ness: This second value calculates cell
number based on the average thick-
ness of the tissue. You would use this
if you measured section thickness at
every sampling site (or periodically dur-
ing counting) and it will calculate the
counts based on the mean thickness
throughout the tissue. This is the *MOST
accurate* of the cell estimates and we
recommend that our customers mea-
sure at every site and use this estimate
for their data analysis.
Estimated total number by weighted
thickness: This third value calculates
cell number by taking into account very
wavy tissue. So if you noticed that there
was a lot of variation between sampling
sites in terms of section thickness, you
should use this cell count estimate.
You can also see the various CEs by
clicking on them. Clicking on Planim-
etry will display the volume and area for
the structure.
Software Bugs: (Save Often)
• If No Live Image
If no live image appears check camera activ-
ity LED on top of camera body; if it is green
it is on but not talking to SI or NL. If Green
do this: (Amber icker = Active)
1. Close software
2. Turn o Master switch
3. Restart Computer
• If Software locks up
1. Turn o master switch
2. Restart Computer
3. Turn on Master switch
4. Login to clear logbooks; depending on
the crash, ITG logbooks may not close until
a user has logged in to the computer.
• Before You Leave:
Close the software and restart the computer
instead of log out. This will ensure all back-
ground processes are stopped, improving
stability for users after you.

Start-up . . . . . . . . . . . . .
1 . . . . . . . . . Turn ON Master Switch
2. . . . . . . . . . . Log on to Computer
3. . . . . . . . . . . . . Load Software
Optional . . . . .Turn ON X-Cite Hg Lamp
Align Optical Components . . . .
1. . . . . . . . . . .Koehler Illumination
Calibrate System . . . . . . . . .
1. . . . . . . . . . . . . Grid Tune Lens
2. . . . . . Parcentric Parfocal Calibration
Run Experiment. . . . . . . . . .
1. . . . . . . . . . . . . .Set up Subject
2. . . . . . . . . . Set scope to Low Mag
3. . . . . . . . Trace Region(s) of interest
4. . . . . . . . . .Set scope to High Mag
5. . . . . . . Dene Counting Frame Size
6. . . . . . . . . Dene SRS Grid Layout
7. . . . . . . . . Dene Disector Options
8. . . . . . . . Save Sampling Paramters
9. . . . . . . Select Markers for Counting
10 . . . . . . . . . . . . .Count Objects
11 . . . . . . . . .View Sampling Results
Save Files . . . . . . . . . . . . .
1. . . . .Save to local machine-(often)
2 . . . . . . . Copy Files Server (Zeus)
1. Koehler align optic train.
2. Deect light to camera with push
pull rod located above binocular vertical
control knob.
3. Start StereoInvestigator. Select Imag-
ing > Live Image (Ctrl-L)
4. Click with mouse inside image frame
to set reference point and to improve
performance of mouse when hovering
over the display area.
5. Select Tools > Video > Camera Set-
tings... to bring up Camera Settings win-
dow to help with setting exposure Gain
and other camera specic options.
6. Adjust Exposure and white balance
using More... button in the Camera Set-
tings window, another window will pop
up with the basic Olympus PictureFrame
software interface “Auto exposure” and
“AWB” auto white balance are the two
most important functions. Ensure to
select an area for white balance in the
Olympus PictureFrame “live window.”
With StereoInvestigator
Acquire Images
7. Create a ateld (optional)
Find a clear area for bright eld, or even-
ly uorescent eld for epi-uorescence;
uorescence dye slides are available on
request.
Capture image: Imaging > Acquire
Image. Set the captured image as a
background image:
Flateld for Brighteld: Imaging >
Set to Brighteld Background Image
or
Flateld for Fluorescent: Imaging >
Set to Fluorescent Background image
Tell software to subtract background
image during acquisition: Imaging > En-
able Background Correction.
8. Acquire images: Imaging > Acquire
Image. The acquired image will replace
the live image remember to select Imag-
ing > Live Image (Ctrl+L) to return to live
viewing of sample.
9. Save Images select File > Image Save
or Image Save As to save captured im-
ages.

Acquire Images Objective List
All require 0.17mm coverslip unless noted
2.5x . . . . . . . .Plan-Neouar1
NA . . . . . . . . . . . . . . . . . 0.075
WD(mm) . . . . . . . . . . . . . . . 9.5
Depth of Field (560nm) . . . . . . 139mm
Part # . . . . . . . . . . . . . . . 440310
5x . . . . . . . . .Plan-Neouar1
NA . . . . . . . . . . . . . . . . . 0.075
WD(mm) . . . . . . . . . . . . . . . 9.5
Depth of Field (560nm) . . . . . . 31.1mm
Part # . . . . . . . . . . . . . . . 440310
10x . . . . . . . . .Plan-Neouar1
NA . . . . . . . . . . . . . . . . . 0.30
WD(mm) . . . . . . . . . . . . . . . 5.6
Depth of Field (560nm) . . . . . . 8.69mm
Phase . . . . . . . . . . . . . . . . Ph-1
DIC . . . . . . . . . . . . . . . . . DIC II
Part # . . . . . . . . . . . . . . . 440331
20x . . . . . . . Plan-Apochromat3
NA . . . . . . . . . . . . . . . . . 0.80
WD(mm) . . . . . . . . . . . . . . 0.55
Depth of Field (560nm) . . . . . . 1.34mm
DIC . . . . . . . . . . . . . . . . . DIC II
Part # . . . . . . . . . . . . . . . 440640
40x OIL . . . . .Plan-Apochromat3
NA . . . . . . . . . . . . . . . . . . 1.4
WD(mm) . . . . . . . . . . . . . . . .19
Depth of Field (560nm) . . . . . . 514nm
DIC . . . . . . . . . . . . . . . . .DIC III
Part # . . . . . . . . . . . . . . . 440865
50x EC Epiplan Air. . . EC-Epiplan4
NA . . . . . . . . . . . . . . . . . . 0.7
WD(mm) . . . . . . . . . . . . . . . .1.1
Depth of Field (560nm) . . . . . . 1.27mm
Coverslip . . . . . . . . . None required
DIC . . . . . . . . . . . . . . . . .DIC III
Part # . . . . . . . . . . . . 422070-9960
63x OIL. . . . .Plan-Apochromat3
NA . . . . . . . . . . . . . . . . . . 1.4
WD(mm) . . . . . . . . . . . . . . .0.19
Depth of Field (560nm) . . . . . . 561nm
DIC . . . . . . . . . . . . . . . . .DIC III
Part # . . . . . . . . . . . . . . . 440762
100x OIL . . . .Plan-Apochromat3
NA . . . . . . . . . . . . . . . . . . 1.4
WD(mm) . . . . . . . . . . . . . . .0.17
Depth of Field (560nm) . . . . . . 514nm
DIC . . . . . . . . . . . . . . . . .DIC III
Part # . . . . . . . . . . . . . . . 440780
Objective Notes:
1.) Plan-Neouar: very good quality-correct-
ed for spherical aberration and corrected
for chromatic aberration in red and blue
wavelengths.
2.) Achroplan: good quality-corrected for
adequate eld atness (spherical aberra-
tion) and chromatic aberration in red and
blue wavelengths to a lesser degree than the
Neouar. A compromise for special applica-
tions like long working distance.
3.) Plan-Apochromat: excellent quality cor-
rect for spherical aberration and corrected
for chromatic aberration in red, blue and
green wavelengths.
4.) EC Epiplan: Enhanced Contrast Univer-
sal objectives for materials microscopy
allowing brighteld, dierential interfer-
ence contrast and polarization methods.
HD versions provide darkeld capability in
addition.
Acquire Image Stacks (Z-Series).
1. Select Imaging > Acquire Image Stack
Spatial Calibration:
Olympus Microre color CCD . . .
2.5x Bin 1 . . . . . . . . . . 2.8 mm/pixel
5x Bin 1 . . . . . . . . . . . 1.45 mm/pixel
10x Bin 1 . . . . . . . . . . 0.73 mm/pixel
20x Bin 1 . . . . . . . . . . 0.36 mm/pixel
40x Bin 1 . . . . . . . . . . 0.18 mm/pixel
50x Bin 1 . . . . . . . . . . 0.14 mm/pixe
63x Bin 1 . . . . . . . . . . 0.11 mm/pixel
100x Bin 1. . . . . . . . . . 0.07 mm/pixel
Original images found in: \stereology work-
space\Tutorials\Calibration Images
2. Select Options... then Set top and
bottom, this will change the appearance
of the Image acquisition menu allow-
ing selection of a focus point above the
sample and a focus point below. Set the
top of the stack and the bottom of the
stack.
3. Set the distance between images to
any reasonable number from .1 mm to
With StereoInvestigator
2mm depending on the objective.
4. Set the delay between images to 750
ms, this will allow the stage time to settle
before the next image is acquired.
5. Acquire image stack.
Multichannel acquire with bring up ad-
vanced options for uorescence imaging.
6. Save image stack.
Setting delay to at least 750ms will re-
duce stage chatter during image acquisi-
tion.

Saving les
1. Save to Local Machine. --Default
You may save les to Workspace (E:\Workspace\”username”). A folder was created
there at login for each user.
2. Before leaving, save to Network Share -- “username” on ‘ITG File Server (zeus.itg.
uiuc.edu)’(Z:)
Most users opt to save the data to their network share on our central server (named
Zeus). The server share is represented as the‘Z:\’ drive in the windows environment.
ITG is equipped with gigabit ethernet, and saving to the Z:\ drive is relatively fast.
Also, this permits easy access to the data from any computer in the world using
secure le transfer protocol (sftp). Instructions on accessing your network share
remotely are available at http://www.itg.uiuc.edu/help/datahandling/userhelp.htm.
Saving as TIFFs (.tif )
Single images, z-stacks, multi-channel images will be saved as a series of TIFF images
that can be viewed in other programs (Adobe Photoshop, ImageJ, Imaris, Image Pro
Plus etc…).

References
Calibration Standards
Calibration Grid Slide from Micro Brighteld:
http://www.mbfbioscience.com
Fluorescent Reference Slides from Micros-
copy Education:
http://www.microscopyeducation.com
Olympus
http://www.olympusamerica.com/seg_sec-
tion/seg_home.asp
--Olympus Microre color CCD
• CCD Array: 1600 x 1200 Pixels
• CCD Size: 14.8 mm diagonal
• Pixel Size: 7.4 microns square
• Architecture: Interline CCD, Progressive scan
(noninterlaced)
• Well Depth: 40,000 electrons
• Read Noise: 20 electrons rms
• Dark Current: 0.3 nA/cm2
• Dynamic Range: 60 dB
• Exposure Time Range: 500 microseconds - 60
seconds
• Gain Range: 1x - 16x
• Bit Depth: 12-bit Monochrome
Commercial anti-fade mounting media
• ProLong Gold Antifade Mountant:
http://www.invitrogen.co.jp/products/pdf/
mp36930.pdf
• Vectashield Antifade Mounting Medium:
http://www.vectorlabs.com/VECTASHIELD/
VECTASHIELD.html
• VECTASHIELD HardSet Mounting Medium:
http://www.vectorlabs.com/products.details.
asp?prodID=1483
Fluorescence Microscopy Books/Papers
Allan, V., Ed., Protein Localization by Fluo-
rescence Microscopy: A Practical Approach,
Oxford University Press (1999).
Andree, M. and Pinkel, D., Eds., Introduction
to Fluorescence In Situ Hybridization: Prin-
ciples and Clinical Applications, John Wiley
and Sons (1999).
Herman, B., Fluorescence Microscopy,
Second Edition, BIOS Scientic Publishers
(1998).
Michalet, X., Kapanidis, A.N., Laurence, T.,
Pinaud, F., Doose, S., Pughoet, M. and
Weiss S., “The power and prospects of
uorescence microscopies and spectrosco-
pies,”Annu Rev Biophys Biomolec Struct 32,
161–182 (2003).
Murphy, D.B., Fundamentals of Light Micros-
copy and Electronic Imaging, John Wiley and
Sons (2001). Molecular Probes.
Spector, D.L. and Goldman, R.D., Basic
Methods in Microscopy, Cold Spring Harbor
Laboratory Press (2005).
Yuste, R. and Konnerth, A., Eds., Imaging in
Neuroscience and Development: A Labora-
tory Manual, Cold Spring Harbor Laboratory
Press (2004).
Stereology References
Online Assistance:
http://www.mbfbioscience.com/live-support/
FAQ:
http://www.mbfbioscience.com/faqs/
Stereology References:
http://www.mbfbioscience.com/stereo-investigator-bibliography/
Chroma Filter Sets for AxioImager A1
Excitation Wheel
(1) Blank
(2) Dual Cy2/Cy3
(3) 350/50
(4) 402/15
(5) 490/20
(6) 555/25
(7) 645/30
(8) Blank-blocked
(9) Blank-blocked
(10) Blank-blocked
Emission Wheel by Camera
(1) IR lter
(2) Dual Cy2/Cy3
(3) 455/50
(4) 525/36
(5) 605/52
(6) 705/72
(7) Blank-blocked
(8) Blank-blocked
(9) Blank-blocked
(10) Blank no IR lter
Filter Cubes/Dichroics
(1) Transmitted cube with polarizer
(2) Cy2/Cy3 lter cube for viewing
through binoculars
(3) 3channel Dichroic --Camera only
(4) 4 channel Dichroic --Camera only
(5) Blank
(6) Reected Brighteld cube
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews, Urbana IL 61801 USA
Phone: [phone] FAX: [phone]