Olympus Spinning Disk - Photomanipulation unit

Start the laser(s) you would like to use by turning the key to "ON".

The 355nm laser is next to the monitor. While the others are bellow the microscope table.

Press the "Start" button when it turns red.

Switch on the controller box under the microscope table.

Optional: switch on the 405nm laser line by turning the key. Press the start button when it turns red.

Optional: switch on the 473nm laser lines by turning the key. Press the start button when it turns red.

Start CellSense

Optional: For a triggered experiment use a template from "Favorite Templates" --> eg. 488 Stim 355.

Otherwise choose and activate the appropriate observation method that includes "STIM" (e. 488nm STIM405).

In this example an image is taken before stimulation and one after.

Localize and select the "Rapp SysCon to CellSens" tab.

"Rapp SysCon to CellSens"

Here you can activate the image feed to the SysCon software once it is opened.

If the tab is not available in your profile, right click on the top of the "Stage Navigator" window.

Select the appropriate option - "Rapp SysCon to CellSens".

Start SysCon software.

Log in using your profile.

If you haven't created a profile yet, follow the next step.

If this is your first log in, create your own user profile.

Choose "Existing user" - "ZMB User 2021".

Create a new user name.

In SysCon: If you are using the 355nm laser you will need to calibrate the system.

Choose the "Motion Control" option on the lower panel.

Press "Calibrate" on the right side of the Motion Control panel.

Once the Calibration is finished it will turn green.

Choose the appropriate objective by hovering over it to display the description. Click on the appropriate one (on the image e.g 40x).

In CellSense: Start Live mode using the appropriate Observation method e.g. "488 Stim 355".

In CellSense: start "Feed Image".

InSysCon: Start Acquisition.

You can control laser intensities here. The 355nm laser line will be controlled in this panel, while the 405nm and 473nm can be modulated within the experiment/sequence.

Additionally you can add / exchange or remove the ND-2 and ND-3 filters. Please make sure you check which filter is in position depending on the power outputs you need.

e.g. for ablation you should start with ND-2 inserted and remove it if you need higher powers to induce damage.

SysCon top right: Open the shutter of your laser of choice.

KEEP LASER STATUS OFF. This will be controlled by your experiment. Otherwise you will already start frying your sample!

Use the "Click And Fire" mode to test your settings or start your experiment.

Choose it under the UG 42 panel (lower right).

Choose the duration of the stimulation and appropriate light source.

355nm laser intensity has to be modulated under the Laser window or by adding/removing the ND filters (see previous step).

In the editor "Point" is pre-selected and appears as a circle in the "Live acquisition" image.

Aim at your region of interest and press left mouse button to "Fire".

If you want to choose different shapes/patterns or define a (triggered) sequence choose "Sequence Mode" under the UGA-42 panel.

To create a new sequence choose "Add".

Make sure you have "Overlay" selected so you can visualize the inserted objects.

Select the desired geometrical object from the panel.

Click into the camera image to position the square. Keep the mouse button pressed to adjust the size.

A corresponding item will appear in the timeline as a bar, after releasing the mouse button.

Use this button to fit the whole sequence into the timeline.

Select the object on the timeline to adjust the appropriate light source and exposure settings.

To create a new object repeat the previous points.

Further objects will appear in the timeline and can be moved as desired.

Make sure you have selected the apropriate observation method in CellSense and that the laser shutter is open.

Upload the sequence.

You will see the progress bar now at "100%" in the sequence.

To play immediately your sequence choose "Button-Click".

Select the number of sequence cycles (Runs). If 0 it will loop until you click the stop button.

Play your sequence.

Define your sequence in CellSense including a TTL pulse .

In SysCon: You can choose one of the available sequence templates as a starting point (e.g. TTL in and out) .

In SysCon: It is possible to create TTL pulses by right-clicking into one ’Output’ line on the timeline and choosing "Add TTL Pulse" from the context menu.

In SysCon: It is also possible to add TTL breakpoints via ’Add Breakpoint’ in the context menu.

These breakpoints can be positioned between objects in the timeline and appear as yellow vertical bars drawn over all timelines. The sequence will stop at each breakpoint until a TTL pulse is recognized at ’Input 1’ of the UGA 42 firefly.

In SysCon: Upload and run your sequence.

In CellSense: Start your experiment including a TTL pulse and wait option.

For more details please ask ou staff.