Zeiss Axio Observer (Schlieren) - Part 2: Basic Acquisition

Start Zen software via desktop shortcut

Software will crash when logging in for the first time. Just kill Zen by the Task Manager and restart software

Change to "Acquisition" tab

Click on Experiment drop down menu

The "Quadband" profiles will only switch exposure light when recording muliple channels. It is significantly faster compared to the "Singleband".

The "Singleband" profiles will change excitation filter cube when recording multiple channels (requires ~1 sec for a switch). The filters can be exchanged and are slightly more narrow than the Quandband filter. Do not use this setting for live imaging.

The individual Quadband/Singleband profiles differ only in the transmission light illumination settings.

BF = Brightfield; Ph1/Ph2 = Phase Contrast; DIC = Differential interference contrast. The optimal transmission light setting depends primarily on the used objective.

The SpectraX_ profiles are write protected. You can make your own templates to save all your settings. Click on the menu button and select "Export"

Save your template in the following folder location in order to make it appear in the drop down menu: Z:\"your core login"\Carl Zeiss\Zen\Documents\Experiment Setups

This microscope uses a custom made light source that is not fully supported by the Zen software. Other approaches to set up the channels like using the "Smart Setup" will not work!

Load slide on stage (coverslip faces down towards objective).

Double click the channel you want to use for finding the focus (e.g. Alexa Fluor 488).

Click the "Live" button to start acquisition (you should see light coming out of the objective).

The image typically stays dark because the Display Adjustments are not set correctly. Ensure you have ticked the "Auto" box above the histogram

You should see now at least some background noise in the image window.

Use the focus wheel on the touch panel to bring your cells in focus

While the "Live Acquisition" is running you can double click on other channels to check signals

Only channels with ticked boxes will be recorded. Click on "Snap" to take image.

New images will show up as tabs above the current image. Recordings can be saved by right click on the tab and selecting "Save as CZI file"

You may need to adapt display adjustments or click the Auto button

Manual saving can be very tedious. Tick the Auto Save box to save images automatically in a defined location.

Single images can be directly saved on the file server in your data folder

More complex experiments like tile images, time lapse acquisitions or automated recordings should be saved locally in D:\Data\Temp\"your name". Do not forget to transfer them to the file server after the acquisition is finished. Files in the local temp folder will be automatically deleted after 1 week.

The default 100 ms exposure time typically works quite well for the 63x oil objective.

When switching to a lower magnification objective it is likely that one needs to change the exposure time settings.

The microscope is equipped with a 16 bit camera which enables a huge dynamic range. It is not advisable to increase the exposure time to fill the complete histogram.

Examples taken with the 20x objective

10ms exposure generates a very noisy image

100 ms exposure still contains some noise

500 ms looks fine

5000 ms does not improve the image quality even though the signal appears to fill the complete dynamic range. This setting is just very impractical to work with because the recording becomes very slow and you may experience bleaching

The high resolution camera in combination with the 16 bit recording results in a large default image size of ~8 MB per channel.

You have two options to reduce the image file size

Option 1: Change the Binning settings under "Acquisition Mode".

Changing to 2x2 or 4x4 binning will increase pixel size and reduces the resolution within your image. If this will be applicable depends on your experimental question.

Option 2: Crop the camera field to record a smaller region of interest (ROI). The pixel size won't be affected. This is very useful if you are anyway interested in single cells but won't help for recording tile images.