Zen Software For The Zeiss Axioobserver: Standard Workflow

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ZEN software for the Zeiss AxioObserver: Standard Workflow I. Choose the pre-set Experiment from the Experiment Manager II. Select channels for imaging under Channel tab III. Go to ‘Live’ and adapt focus, exposure time etc Possibility: if having a sensitive sample take a Snapshot and adjust display settings to check the final image ‘quality’ and re-adjust if necessary Display tab: - min/max function will show the histogram optimally (relevant info 'normalized') - gamma function will correct for background (diminish low grey level impact while keeping the higher grey level intensities) To visualize single channels, activate ‘Single channel’ option in Dimension tab IV. Tiles (Optional) 2 main application workflows: 1. 'Statistic': Sample carrier option allows to automatically distribute regions and positions within selected wells 2. 'Precise': Single position option where positions can be added manually without sample carrier exactly on the place of interest 1. Using Sample carrier option 1.1. Select and/or create an appropriate layout with ‘Copy and edit’ function (under little 'settings' wheel down right or the right click on the carrier of interest. First time using a carrier layout one needs to create a user copy and calibrate it. For the future use this will be saved in the user profile.) 1.2. Rename the carrier and choose custom Location mode for the reference point. Place the yellow cross at the recognizable area of the sample (for example top of the 1A well), confirm 1.3. CALIBRATE: - Show full light path: make sure that Shutter is open, signal is directed to camera. - Switch on transmitted light at the top of the window and adjust the intensity on the wheel on the microscope (lower front)  Next choose ‘Search one reference point’ Next  Find the position of 'yellow cross' in reality under the microscope Set current XY  Finish In the main window go to Advanced setup: – choose the channel you want to use to define the position with (if applicable transmitted light recommended to save sample from bleaching) – Using the 'Carrier’ tab in the advanced setup one can go in the well of interest and/or select different wells and use the 'Position setup' to define positions using Carrier/Location/Array 2. Using Single position option 2.1. Find the position of interest and add it to saved positions in the main Tiles window V. VI. VII. VIII. Decide if one uses Time laps or not and then check the Focus strategy. If using the Tiles option before starting experiment readjust XYZ positions individually, if necessary for each channel Readjust the exposure time where objective, region etc changes Start the experiment.