Visitron Spinning Disk - Acquire an Image in XY and Z

Acquire: control acquisition parameters as illuminations, exposure, gain, binning e.g., adjustments of z-stacks, timelapse or multi area detection

Control Panel: controlling LED and/or laser intensities.

Overlay: set which acquired images should be overlayed for display

Histogram: display of the intensity distribution of the active image

Image Display: option for auto scaling, intensity values, adding annotations to image e.g.

Processing: there are different processing options available within VisiView software

Macros: most used functions and toggling between objectives.

Please note, the interface can slightly differ between users (where each window is located, or if active e.g.). Windows can be activated/opened via the menu bar on top, and can be rearranged via drag and drop.

Have your sample mounted and focused as described in Start-up guide.

Select a "Wavelength/Illumination" (here e.g. EpiDAPI) within the "Acquire" window.

Adjust Camera "Exposure" time and "EMCCD gain".

Adjust LED/Laser intensity within the "Control Panel".

Click "Show Live".

Check "Histogram" and "Image Display" to optimize imaging parameters (as exposure, gain and intensity). Avoid any saturation.

Camera(s) run in 16 bit mode, so your max should be lower than 65535.

"Binning" and "Live Bin" - Binning can be used to increase sensitivity in low-light conditions, read-out times get shortened, but resolution will also decrease. "Binning" - any adaptions here will be applied to the final image acquisition. "Live Bin" - only applied during live mode.

Depending on which tabs are active you can decide if you wanna save your data automatically or manually. For some options data must be automatically saved.

Within the "VisiView" software in "Acquire" window define where your data should be saved:

Define "Base File" name.

Define Storage location via "Directory".

There are two options to save your data:

Choose "automatic_transfer (X:)" and generate a new folder. Details can be found in the following guide "Visitron spinning Disk - Datamover".

Save directly to network on "files.core.uzh.ch (Z:)". Details can be found here.

Finally press "Ok".

The system is equipped with 2 EMCCD cameras.

Imaging can be performed either with one camera (CAM1) or with both (CAM1 and CAM2) using the TwinCam module, depending which wavelength setting is chosen.

The TwinCam module allows for fast and sensitive simultaneous acquisition of two-color (live) samples.

Preset illumination selections for one camera approach - Conf~, Epi~, and BF drop down.

Preset illumination selections for dual camera (TwinCam) approach - TwinCamDual~ and TwinCamSingle drop downs.

For one camera approach please follow the next steps.

For dual camera approach using the TwinCam module please refer to the following guide "Visitron Spinning Disk - Dual camera TwinCam" (guide is still in preparation).

Within the "Acquire" window select "Wavelength" tab and activate "Wavelength Series". The "*" represents if this series is activated.

Define how many channels you would like to image (here 4 e.g.).

For each channel ("Current") select an "Illumination" wavelength.

Adjust your imaging settings (exposure, gain, LED/laser power) for each channel by selecting it first under "Wavelength" and then click "Show Live".

Saturation can be displayed in any wished Pseudocolor by checking the box "Show Saturation".

Once satisfied with your settings for each channel click "Stop Live".

You can define if "Exposure/Gain" should be same for each wavelength by marking "Same Exposure/Gain ..." if applicable. Further re-adjustments might be required.

Once you are satisfied with your settings, click on "Sequence" to start acquisition.

Depending whether you selected "Save Sequence to Disk", the data will be already saved or not.

To observe an overlay of the acquired channels:

Select how many channels you wanna overlay.

Select which ones in the drop down menus.

Click "Show".

Here it shows "Hide" instead of "show", as the overlay is already open.

Select "Z-Series" tab and activate "Z-Plane series".

To configure which device be used go to "Configuration" and select "Focus".

Within the opened window "Focus Configure" select your "Focus Device" from the drop down menu.

Virtex - ZFocus#2: Fast Piezo stage - the travel range of this device is 350um. Make sure the Piezo is in the middle of the travel range (175) before you start your experiment.

Nikon - Focus - full travel range of the stage, so used for thicker samples (more than 350 um).

Select if you wanna "Acquire Wavelengths at each Z" or acquire "Z Series for each Wavelength".

Click "Ok".

Click "View Home" and focus.

Define now your top and bottom limits of your sample:

Click "View Top Offset" and adjust the value that you reach the top of your sample.

Click "View Bottom Offset" and adjust the value accordingly for the other side of your sample.

Stop Live View when satisfied.

Define your step "Size" (unit = um). Z-stack direction can be alternated by adding a "-" in front of your defined Step-Size.

Click "Sequence" to acquire a z-stack.

One can toggle through the different z-layers using the keyboard arrow keys up/down.

Alternatively you can also choose to define a centered range.

Check "Centered Range".

Define your volume size.

Make sure that your current focus is in the middle of your sample.

Define your step "Size" (unit = um). Z-stack direction can be alternated by adding a "-" in front of your defined Step-Size.

Click "Sequence" to acquire a z-stack.