Zeiss AxioObserver User manual

Zeiss AxioObserver with ApoTome
Quick Start User Guide (AxioVision4.8)
LSU Health Sciences Center-Shreveport
Research Core Facility (RCF)
Microscopy

RCF Microscopy Manual 5/2/2017
Authors Cory Nook and Chaowei Shang
Page 2of 19
Table of Contents
1
Start up the system……………………………………………………………………………………………………..
Page
3
2
Touch screen controller (TFT DISPLAY)
Page
5
3
Mount and view the sample through the microscope ……………………………………………….
Page
7
4
Start up the AxioVision software….……………………………………………………………………..........
Page
8
4.1
Experiment settings……………………………………………………………………………………………….
Page
9
4.2
Apotome Settings………………………………………………………………………………………………………
Page
10
4.3
Channel Creation……………………………………………………………………………………………………….
Page
11
4.4
Setting Exposure
Page
12
5
Acquiring and saving an experiment …………………………………………………………………………..
Page
13
5.1
Acquire………………………………………………………………………………………………………………………..
Page
13
5.2
Save and Export……………………………………………………………………………………………………….
Page
13
6
Z-stack ………………………………………………………………………………………………………………………
Page
15
6.1
Adding a Z-Stack to Your Experiment…………………………………………………………………………..
Page
15
6.2
Setting the Limits of the Z-Stack………………………….…………………………………………………….
Page
15
6.3
Z-Stack Settings……………………………..…………………………………………………………………………..
Page
16
6.4
Z-stack Cut View…………………………………………………………………………………………………………
Page
17
7
Shutdown……………………………………………………………………………………………………………………
Page
18
8
Technical Issues and Errors….……………………………………………………………………………………..
Page
19

RCF Microscopy Manual 5/2/2017
Authors Cory Nook and Chaowei Shang
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If the room lights are not on when you arrive to use the Axioobserver, DO NOT TURN THEM ON. Please be courteous of
those using the other microscopes, since they may have light-sensitive slides, dishes, or plates on the microscope stages,
which could be damaged by the room lights. This is particularly the case with the Leica confocal, since some Z stacks can
take over half an hour to complete. Use a torch if needed.
Check whether the floating stage is functional. If not, please contact the Research Associate. Make sure the
table is clean. No food or water is allowed in the microscope room. Carefully take off the microscope cover
and put it in the white storage cabinet. Check whether the holder is properly in place.
Please check the objective turret has the 10x objective in place. This is a safety precaution. If the 10x objective
is not in place, please clean the current objective and manually switch it to the 10x objective. To manually
switch objectives, push on the teeth of the objective turret in either a clockwise or counter clockwise fashion
as shown adjacently in the boxed region.
1. START UP THE SYSTEM
Please sign in on the login sheet. The format for the date is MM/DD/YYYY, eg. 05/25/2015. Write legibly for
this is how the staff knows who has been using the microscope. Check in and Check out time is written in 24
hour format, e.g. 1pm is 13. The format for writing the bulb time is ###h ## m, eg. 100h 25m. (You will not be
able to write the bulb time until you turn on the power supply.)
Our system has the startup labeled
numerically with yellow stickers.
First, turn on the mercury lamp power source.
The green power light and the green lamp
light will come on. (This is where you record
the bulb time.) If the lamp light indicator does
not light up, please contact Core facility staff.

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Authors Cory Nook and Chaowei Shang
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Once turned on, the lamp should remain on
for a minimum of 30 minutes. Do not turn the
lamp off if another user is scheduled within 2
hours after your session.
Secondly, turn on the Power Supply 2 by
flipping the switch which will have a green
light turn on.
Third, you will need to go in between the
microscope and the power supplies to press
the button below the yellow sticker with the
number 3. See image to the right. This will
turn on the TFT Display, shown adjacently, and
please wait for this display to fully load before
proceeding.
Lastly, turn on the Apotome power supply
labeled with a yellow 4 sticker.
The computer should be on and logged in with your LSUHSC ID and password. Make sure the domain is set to
LSUMC-MASTER, selected from the drop-down menu. If you only wish to access documents/files, then login
and do not turn on the mercury lamp power supply or the other buttons. Please indicate this on the sign in
sheet.

RCF Microscopy Manual 5/2/2017
Authors Cory Nook and Chaowei Shang
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2. TOUCH SCREEN CONTROLLER (TFT DISPLAY)
To change the position of the microscope stage, and
thus your sample, you will need to use the adjustment
knob and joystick attached to the TFT display. The
adjustment knob, on the right side of the TFT display,
has two “wheels” for different adjustments and controls
the Z-direction of movement. The larger wheel is used
for course adjustment while the smaller one is used for
fine adjustment. The joystick behind the TFT display will
control the X and Y direction of the stage. The degree of
which the stage will change will depend on which
objective is in the light path.
The image to the right displays the Home screen and we
will go over useful information, in the center, that can
assist you in imaging.
Objective: Description of the current objective in the
light path along with N.A.
Resolution: How fine of detail the current image will
appear
Optovar: A tool that is part of the system that can be
used like a magnifying glass. This should always read 1x
and should be changed to this in compliance with
Apotome use.
Total Magnification: The number of times the image has
been “expanded” compared to the actual size of the
image
HAL: The current voltage of the halogen bulb, this is only
a concern for brightfield imaging.

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Authors Cory Nook and Chaowei Shang
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To select an objective, you can either move the
objective manually, or using the TFT display. We
recommend using the TFT display for it is easier to
select objectives. On the home screen displayed, push
the icon labeled Microscope which will pull up a new
screen.
The adjacent picture shows the screen for the
Microscope control panel on the TFT display. Make
sure that the Objectives tab is selected as it will be a
light blue colored compared to the rest. Also, ensure
that you are on the Control display by checking to see
if the Control icon on the sidebar is white and not
blue. An icon is selected if it is white and not selected
icons will appear blue, as shown to the right. The
Objectives displayed tell you which one is an air
objective versus an oil objective. If you need to switch
between different objectives that use different
immersion, the software and TFT display will inform
you and confirm that you are ready for the next
objective.
Reflector: Displays which position of the reflector turret
is in the light path and what type of cube is in that
position.
Condenser: Describes which type of brightfield imaging
is in place along with the N.A.
VIS: Displays how much of the light from the sample is
being sent to the eyepiece
Sideport: Displays how much of the light from the
sample is sent to the camera/computer

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To illuminate your sample with fluorescent light,
press the header icon labeled Reflector to pull up the
fluorescent filters. On the right sidebar, make sure
that the Transmitted light (TL) Illumination is off and
that the Reflected Light (RL) Illumination is on.
Filter choices are labeled as, on the TFT display:
34 BFP (EX 390/20, EM 460/60) –for DAPI, Hoerchst, AMCA
38 HE GFP (EX 470/40, EM 525/50) –for GFP, FITC, and
prevents red emission bleed through
31 AF 568 (EX 565/30, EM 620/60) –for Cy 3, Texas Red
17 AF 488 (EX 485/20, EM 540/25) –for GFP, FITC, and
prevents red emission bleed through
50 Cy 5 (EX 640/30, EM 690/50) –for Cy 5 and far red dyes
3. MOUNT AND VIEW THE SAMPLE THROUGH THE MICROSCOPE
If you are using an immersion objective, place a VERY small drop of the appropriate immersion fluid on the
objective face, or the coverslip. Be sure to only use the immersion fluids located next to the microscope. If you
accidentally use the wrong fluid, please contact staff right away. If you are using a slide, insert it coverslip
down into the specimen stage template. Below is a table of our current objectives, imaging medium, and
brightfield capabilities.

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Obj
Objective
Medium
Grid
10x
EC Plan-Neofluar 10x/0.3 DIC I
Air
L
20x
Plan-Apochromat 20x/0.8 DIC II
Air
L
40x
LD Plan-Neofluar 40x/0.6 Ph2 DIC II
Air
NA*
40x
Plan-Apochromat 40x/1.3 DIC (UV) VIR-IR
Oil
L
63x
Plan-Apochromat 63x/1.4 DIC III
Oil
H
100x
Plan-Apochromat 100x/1.4
Oil
D
The 4 buttons on the template move the support brackets along the track. The template will hold a variety of
shapes. If you need a multi-well plate template, please ask staff.
Start with DAPI to find and focus on your sample. Be aware that fluorescence light is damaging to the eyes.
Avoid looking directly at the lasers. Block the light infront of you if the orange light shield is not strong enough
to block the light.
4. START UP THE AxioVision SOFTWARE
Double-click on the AxioVision icon on the desktop to
start the program.
Make sure your Apotome power supply (labeled with
number 4) is turned on before starting up the
software, otherwise apotome may not work properly.

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4.1 Experiment Settings
The default page for the AxioVision
software is shown on the right. A
majority of commands can be found
in the left side bar labeled
Workarea. You can control filter
cube selection, light path direction,
objective selection, camera settings,
etc.
The image on the right shows a
more detailed view of the options in
the Workarea. The Microscope
selection allows you to adjust parts
of the microscope similar to the
options in the TFT display. All
options with a plus sign to the left
of the icon indicate that a pull down
menu will appear when the plus
sign is clicked on. In order to adjust
the Camera, Apotome, and to work
with Multidimensional Acquisition,
you must select the plus sign next to
the designated icon. The toolbars at
the top of the screen give you short
cuts to features such as light path
selection, filter selection, and
illumination.

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4.2 Aptome Settings
About the Aptome
The ApoTome uses a projected grid along with a
mathematical algorithm to remove out-of-focus
light, similar to the function of the pinhole on a
confocal microscope. When not in use, it should be
pulled out slightly from the microscope. There are
three small grids (L, H, D) that can be used in the
ApoTome; each objective requires a particular grid.
The software will prompt you to change the grid if it
senses that the grid and the chosen objective do not
match. Sometimes, because objectives are retired
and new ones created, the match between
objective and grid may not be reflected in the
software. A Zeiss engineer helped me come up with
the best pairing for objectives and grids that we
have (see below).
When the ApoTome is pushed in all the way, it is
engaged. The grid moves through 3 positions as it
captures one image. When you need to disengage
the ApoTome from the microscope, turn off its
power supply first (Labeled #4), which allows the
grid to return to its original position, then turn in
back ON before you pull it part or all of the way out.
Failure to do so may cause damage to both the grid
and the Apotome. Should the grid become loose
from its niche in the Apotome, let me know, and I
will retrieve it.
From the menu, choose the ApoTome button. A dialogue box
labelled ApoTome will pop up. Choose the second tab, Settings,
and choose the following:
ApoTome: Live Mode –Grid visible
ApoTome: Acquisition Mode –Optional Sectioning
ApoTome Grid –Choose the correct grid for your
objective from the drop down menu
ApoTome Filter –Medium
ApoTome: Averaging (Noise Reduction) –2
ApoTome: Status –this should say Calibrated in a green
field (if not, come get me!)
Obj
Objective
Medium
Grid
10x
EC Plan-Neofluar 10x/0.3 DIC I
Air
L
20x
Plan-Apochromat 20x/0.8 DIC II
Air
L
40x
LD Plan-Neofluar 40x/0.6 Ph2 DIC II
Air
NA*
40x
Plan-Apochromat 40x/1.3 DIC (UV) VIR-IR
Oil
L
63x
Plan-Apochromat 63x/1.4 DIC III
Oil
H
100x
Plan-Apochromat 100x/1.4
Oil
D
*The 40X Air Objective is a Phase Objective and shouldn’t be used with the Apotome.

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4.3 Channel Creation
Before you start an experiment, you can set up a naming system that will
be discussed in the appendix. In the toolbar, click on the icon labeled 6D-
Acquisition.
This will pull up the following window. Next to the Experiment tab, click
on the blue circle icon to select different channels to acquire. This icon
represents different fluorescent and brightfield channels, or multichannel.
If you have a file already saved and want to repeat those settings, you can
go to the Experiment section of the window and select either Load or
ReUse. You can also give a name to the particular experiment group by
editing the Image name in the Experiment section.

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You will see the next screen to adjust different
channels. To add a channel, click on the
numbered icon that has an X through it at the
top of the window and right click on it with the
mouse. To remove a channel, right click on the
number that is surrounded with colour. Select
a channel by left clicking on the numbered
icon. Each channel allows you to define a dye,
colour, and name. The dye choices are given by
Zeiss and are mainly used for meta data.The
Hardware Settings section is what the
microscope changes for each channel to
acquire the image. For example, if the channel
definition is TRITC and you have the
Workgroup: DAPI selected in the During
acquisition part of the Hardware Settings, you
will get an image of dye excitation similar to
DAPI but will be labelled with TRITC. Select the
arrow the left of the Go icon and it will give
you a pull down menu of work group presets to
acquire images. Select the appropriate
Workgroup setting for the given dye. We
recommend that in the After acquisition
setting, that you turn off the corresponding
light source.
4.4 Setting Exposure
Switch from the eyes mode to the camera
mode to allow images to be shown on the
software. (Switch back from camera to eyes
when observing through the eyepieces.)
The Exposure section is used to capture the
images. The given time is how long the camera
will open the shutter and collect photons from
the specimen given the selected filter cube.
The time can be adjusted manually or by using
the measure button to the right. The measure

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button is recommended to not only get the
correct camera settings but can be used to
make sure your sample is in focus. This is
where the user needs to decide which part of
the sample is the most important. That part of
the sample needs to be in focused because not
all channels may necessarily be focused at the
same position. When the measure icon is
clicked a new window will pop up as shown.
Displayed is that current channel in black and
white to show over exposed pixels. Any pixel in
red is over exposed and cannot be used in
quantification data. Also, over exposure for
long periods of time can damage the camera.
When you have adjusted the exposure time
and focused on your image, click on the OK
button. Do NOT close the window via the close
icon in the top right of the window. This can
lead to a “run-time” error and can bleach your
sample if not careful. See the appendix on
“Run-Time Errors” to know how to prevent
sample bleaching.
5. ACQUIRING AND SAVING AN EXPERIMENT
5.1 Acquire
Confirm that you have switched from the eyes mode to the camera mode
After you have completed all of your settings and are ready to capture an image, click Start at the
bottom of the Multidimensional Acquisition Window.

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Click off pseudo colors and right click on the images, a menu containing property will appear.
Adjustment of images can be done under the property settings.
5.2 Save and Export
YOU MUST SAVE ALL IMAGES ON THE E: DRIVE IN A FOLDER
WITH YOUR NAME. DO NOT SAVE ANYTHING ON THE C:
DRIVE. THESE IMAGE FILES WILL BE PERIODICALLY REMOVED
BY STAFF, SO YOU MUST BACK UP YOUR FILES ELSEWHERE.
To save an image, click on the experiment
heading and select File in the toolbar. The
format for the proprietary software is .zvi.
Make sure you save all your images in this
format (when publishing data, many
journals require the original format of
images). You may also export in other
formats but it is recommended to export as
a tiff, later. It is also advised that you save
at regular intervals to protect your images
from an unexpected software crash.
To export files, go to File in the toolbar and
click on Export. An export window will
appear with the base name of the desired
file in the window heading, shown
adjacently. Create a name for the file in the
Base name: section. This will give the initial
name for the files to be exported and the
name of the folder to where the files will
go. The Save in: section gives the file path
where your files will be saved. You need to
make sure that these files are going to the
appropriate folder. The output files at the
bottom of the window tell you how many
images will be produced in the desired
format. If you want a merged image of all
the channels then you will need to select
Generate Merged Image(s) below the
channel selection.
TIFF

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6. Z-STACKS
6.1 Adding a Z-Stack to your Experiment
Acquire a z-stack only after setting up all the imaging
parameters in the steps above. Then click on the Z-Stack tab
in the Multidimensional Acquisition window. You will need to
select the Z-Stack icon in the upper right corner of the tabbed
window.
6.2 Setting the Limits of the Z-Stack
The system acquires a Z-Stack by defining the top of the
sample and bottom of the sample but actually goes in reverse
order. It is recommended to set the positions of the start and
stop points to be past the region of interest. This is to ensure
that your entire region is in the Z-Stack and that none of it is
mathematically taken out.
Make sure that the mode selected, with a green dot, is
Start/Stop and not Center.
You will need to alternate between this tab and the channels
tab. Click on the channels tab and select one of your
channels. Click on measure and use the TFT display Z-
adjustment knob to move the objective turret upwards until
you are past your desired region of interest. Click on Ok and
then click on the Z-Stack tab again. Next, click on the Start
icon, boxed in the figure on the right.
Follow the same steps above only to move the objective
turret downwards until past the desired region of interest.

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6.3 Z-Stack Settings
Define the number of slices or the z-step size. By default, the
software calculates the optimal z-step, or slice thickness,
based on the axial resolution of the chosen objective and
wavelength, and then determines the number of steps. You
may override this by clicking on the button and entering a
different step size or number of steps in the field. It is
recommended to do an odd number of slices, usually, to
ensure that the middle of your sample is not split between
slices. You can also select the Optimal Distance icon below
the slice distance and it will give you the mathematically
determined option that will be on the verge of oversampling.
The default of the Z-Stack is to go through each channel and
image a Z-Stack. You can select the All Channels per slice
option in the Settings section to allow all the channels to be
imaged per slice before the movement to the next part of the
Z-Stack. Personal preference is the reason for why one
option is selected over the other. Imaging one channel per Z-
Stack allows for the channels to have the specifications of the
slice number and distance. However, the overlay of the
channels might not be exactly the same such that the position
of each slice might be different for each channel. This
problem can be overcome with all channels per slice but this
means that the acquisition will take longer.
At the bottom of the window, you may or may not need to
add settings for the microscope to do certain functions before
the experiment begins. This could be as simple as turning on
the fluorescence or activating only one filter position.
Click Start to begin the acquisition sequence. After capturing
a z-series, you will need to click on Z-Stack icon in the Z-Stack
tab to remove Z-Stacks from acquiring in the future.

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6.4 Z-stack Cut View
The Cut View button will create orthogonal views for the Z-stack images. However, the cut view option
overlays images on top of each other and causes over saturation of the stacked images. To adjust the over
saturated images, do as follow:
(1) Turn off the channel coloring, (2) select the Cut View, (3) select Properties and (4) select Best Fit. Do this for all the
channels by selecting the appropriate channel # next to (1).
(5) Finally turn on channel coloring.

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(6) Click create image, then save/export the cut view image.
7. SHUTDOWN
Check the calendar to see if there is anyone signed up to use the microscope after you. The link to the
calendar login is located on the desktop.
If someone is signed up to use the microscope within two hour of when you have finished, do NOT turn off the
power supplies. Instead, exit the software and log out of your user ID. Also, please write down the ending bulb
time on the signup sheet when you sign out, even if you are not turning it off.
If no one else is signed up to use the system for 2 hours, shut the system down in the following order:
Save your image files and export any desired images as .zvi files.
Remove your sample, clean any immersion objectives used, and select the 10x objective in the
software.
Exit the software and log off the computer via the Start menu of Windows
After you are logged off, please restart the computer
Write the elapsed time on sign-in sheet
Turn off the following in reverse start up order:

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oApotome Power Supply (Yellow Label #4)
oMicroscope (Yellow Label #3)
oMicroscope Power Supply (Yellow Label #2)
oHBO (mercury) Lamp Power Supply (Yellow Label #1)
Cover the microscope (except for any hot parts)
8. TECHNICAL ISSUES AND ERRORS
If you are experiencing any technical issues or errors, please save the corresponding file into the D drive under
the folder labeled “Technical Issues and Errors”. After you have saved this file, please fill out an error log
located on a clip board next to the computer. Please contact the research associate as well to ensure the
problem is not overlooked and fixed as soon as possible.
Sometimes you are not able to save a file but encounter an error which cannot be saved. You will have to take
a screen shot which can be accomplished by pressing the “PrtScn/SysRq” button next to the F12 key. You will
then need to open a Paint application located in the Accessories folder of the Programs folder. After you have
opened a blank Paint document, click on “Edit” at the top of the window and select “Paste” from the drop
down menu. This should paste the screen shot and you will be able to save this to the folder mentioned in the
previous paragraph.
Problems that you may encounter are listed below:
If you are able to see cells with the microscope but cannot see anything when you click measure in the
multidimentional acquisition window. Make sure you have switched from the eye mode to the
camera mode.
When the Apotome gives you a message “apotome not found”, it maybe that the software was turned
on before the Apotome and microscope was turned on. Try to restart the software.
When you are given the message “Apotome not caliberated” in red, turn on a laser first (e.g. DAPI), and
apotome may show as “caliberated” in green.
When your image cannot get focused, check if your slides are properly placed in the bracket. (slides
may be pushed up by objectives).
If you find your adjustment sliding bars (under property) are in grey, check if you clicked off the pseudo
color of the image. The adjustment sliding bars are only activated when the pseudo colors are off.