- For the EM-CCD (low resolution, high sensitivity), pull the handle on the spinning disk unit up and put the small plastic box underneath to fix it (see image right).
- For the sCMOS (fast, high resolution), push it down. Hold it with your hand while pushing (see image left).
- Windows login: pick the right user account (Imaging, FRAP or Nanodissection). Nanodissection is password protected, the others are not.
- Launch VisiView. If you have not turned on all lasers, an info message will list inactive lasers being emulated (Obis, Obis-2, and/or Toptica for the 405, 488, and/or 640 nm, respectively). Acknowledge by clicking OK. Likewise, acknowledge Hard drive not found warnings (the startup script will reset the path for saving files to the standard hard drive D:).
- Startup dialog:
- Camera & Spinning Disk: enter the selected camera & SD (W1 or X1, sCMOS or EM-CCD)
- Objective: choose the one you wish to use from the drop-down list
- Yes (recommended) moves the objective to the lowest (safe) position
- No (are you sure?) should only be chosen if you had to restart VisiView during a session
- Keep settings/positions:
- No (recommended) clears any previous stage positions and illumination settings
- Yes should only be chosen if you had to restart VisiView during a session
- After running the startup dialog, the objective should be in the lowest (safe) position.
- If the ESC (Escape) button on the microscope lights green, the focus is locked. This happens if the previous user has lowered the objective turret at the end of the session using the ESC button. Press it and keep it pressed until the green light turns off.
- Launch the webcam in the box with the camera icon on the taskbar.
- Turn on the light in the chamber.
- At 37°C, rotate the collar so that the mark on the objective body coincides with the 0.15-mm scale mark on the collar. On the 40x silicone objective, adjust to the mark halfway between 0.13 and 0.17, on the 60x water to the mark labeled "15".
- If the CO2 insert is not used, it should be stored inside the box (to keep it on temperature) at the front left corner (caution: avoid blocking the travel range of the XY stage). The click-in plates for the CO2 insert should be stored on one of the shelves inside the box.
- Other inserts (standard slide, multiwell) should be kept in the small drawer next to the door. In the drawer, there is also a click-in plate for two 35-mm dishes.
**Important: **when using the click-in plate for two 35-mm dishes, always move the objective down using the ESC button before moving from one dish to the other!
- The Nikon focus turns in the opposite direction as compared with Zeiss microscopes. An arrow (UP) on the focus drives and on the Nikon joystick pad indicates the direction.
- The Z^ button toggles between coarse (fast) and fine (slow) mode. In coarse mode, the focus indicator on the frame lights green. The current mode is also indicated on the LED of the joystick.
- The PFS buttons on the focus drives and joystick control the Perfect Focus hardware autofocus.
- Pressing the button briefly turns it on or off. The PFS indicator on the frame lighting green indicates an active PFS. It blinks if it is off, but detects a surface within its travel range. It remains dark if there is no surface in reach.
- Pressing it and holding it for >1 sec turns the PFS off and removes the dichroic from the light path. The indicator on the frame turns dark. The LED on the joystick pad displays "PFs:OUT".
- The Nikon XY joystick is inactive. To move the stage in XY, use the ASI controller next to the microscope. Pressing the button on the top of its joystick toggles between slow and fast mode.
- To ease finding the focus at startup, click on the Focus Up button in the VisiView toolbar. In the dialog popping up, select the name of the installed stage insert.
- Center the objective below the opening of the stage insert using the ASI XY joystick and click OK. The objective will move up, close to the sample. It will not be exactly in focus, though, and you will have to focus a bit manually. Clicking the Cancel button in the dialog will exit without action.
- To replace the sample once focused, press the ESC button on the microscope frame. The objective turret will move down. All focus controls will be locked and the ESC indicator will light up green. Briefly pressing the ESC button again will move the objective back to last focus position and enable the focus controls (wait until the green light is off). When keeping the ESC button pressed until the green light goes off, the focus lock will be released, but the objective turret will remain in the lowest (safe) position.
#### 9. CO2 & temperature control (optional)
- By default, the system should be running at 37°C. Do not switch off the controller after your session. If you need lower temperatures, please contact the person signed in after you far in advance and ask for their permission, as the temperature will need some time to increase again after your experiment.
- Open the main valve of the CO2 tank by turning it counter clockwise for 1 round (no need to rotate it up to the stop). Close it clockwise once you are done. Don't touch the other valves!
- Power on the CO2 controller (power switch on its back side).
- Place the CO2 cover on the CO2 insert and make sure that the humidifier bottle is filled with enough distilled water (>50 mL; the bottle can be disconnected with two luer connectors). To replenish water (deionized, autoclaved water only), contact the facility staff!
- File format: in the top menu, select Configure → System. In the dialog popping up, select the Acquisition tab. Under Save File, select file format:
Metamorph STK is recommended for all kinds of multidimensional acquisitions.
OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions in the metadata. Check the auto checkbox. VisiView will automatically select the proper OME Tiff subtype (32 bit as *.ome.tif, 64 bit version as *.ome.btf).
