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AxioObserver
Quick Start Manual
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User Manual
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Zeiss AxioObserver Quick Start Manual

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Zeiss AxioObserver with ApoTome
Quick Start User Guide (AxioVision4.8)
LSU Health Sciences Center-Shreveport
Research Core Facility (RCF)
Microscopy
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1 Start up the system……………………………………………………………………………………………………..
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Touch screen controller (TFT DISPLAY)
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Mount and view the sample through the microscope ……………………………………………….
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4 Start up the AxioVision software….……………………………………………………………………..........
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4.1
Experiment settings……………………………………………………………………………………………….
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4.2
Apotome Settings………………………………………………………………………………………………………
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10 4.3
Channel Creation……………………………………………………………………………………………………….
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Setting Exposure
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Acquiring and saving an experiment …………………………………………………………………………..
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Acquire………………………………………………………………………………………………………………………..
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Save and Export……………………………………………………………………………………………………….
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6 Z-stack ………………………………………………………………………………………………………………………
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6.1
Adding a Z-Stack to Your Experiment…………………………………………………………………………..
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Setting the Limits of the Z-Stack………………………….…………………………………………………….
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Z-Stack Settings……………………………..…………………………………………………………………………..
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Z-stack Cut View…………………………………………………………………………………………………………
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Shutdown……………………………………………………………………………………………………………………
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8 Technical Issues and Errors….……………………………………………………………………………………..
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Authors Cory Nook and Chaowei Shang
Table of Contents
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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.
Authors Cory Nook and Chaowei Shang
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.)
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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.
Authors Cory Nook and Chaowei Shang
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.
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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.
Authors Cory Nook and Chaowei Shang
2. TOUCH SCREEN CONTROLLER (TFT DISPLAY)
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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
Authors Cory Nook and Chaowei Shang
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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.
Authors Cory Nook and Chaowei Shang
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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
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.
Authors Cory Nook and Chaowei Shang
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
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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.
Authors Cory Nook and Chaowei Shang
4.1 Experiment Settings
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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
Authors Cory Nook and Chaowei Shang
4.2 Aptome Settings
*The 40X Air Objective is a Phase Objective and shouldn’t be used with the Apotome.
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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.
Authors Cory Nook and Chaowei Shang
4.3 Channel Creation
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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
Authors Cory Nook and Chaowei Shang
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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.
Authors Cory Nook and Chaowei Shang
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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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
Authors Cory Nook and Chaowei Shang
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
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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.
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 Z­adjustment 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.
Authors Cory Nook and Chaowei Shang
6. Z-STACKS
6.1 Adding a Z-Stack to your Experiment
6.2 Setting the Limits of the Z-Stack
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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 Z­Stack 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.
Authors Cory Nook and Chaowei Shang
6.3 Z-Stack Settings
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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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o Apotome Power Supply (Yellow Label #4) o Microscope (Yellow Label #3) o Microscope Power Supply (Yellow Label #2) o HBO (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.
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