Ensure the objective currently on the microscope is the one that should be used for the experiment. When examining or changing objectives, make sure the key on the laser engine front panel is in the OFF state (or the power to the laser engine is turned off)

Turn on the power to the microscope controller, the camera and the laser engine. It will take a few minutes for the laser engine to warm up (in the next upgrade a status indicating light will be added).

• The microscope controller is powered by a 24V output AD/DC adapter.
• The laser engine is powered by a 24V output AD/DC adapter.
• The camera is powered by a 12V AD/DC adapter.

Enable the laser engine by pressing the on button in the front panel of the laser engine (it will light up) and switch the key to ON position.

Launch the software by double clicking the launcher on the desktop. Choose the launcher that matches the objective magnification.

Load the sample. Make sure the microscope is not in the “Live” mode so that illumination is off. One can switch the key to the OFF location on the laser engine as extra caution.

Move to the well you want to image with the Well Selector widget (by double clicking the well you want to go to).

Select the first channel you want to use for adjusting focus and click Live.

Adjust focus using the focus knob on the joystick. Counter-clock wise movement brings the objective closer to the sample. For fine adjustment, use the top sensitivity adjustment knob to reduce input sensitivity (8 is most sensitive and good for coarse adjustment and smaller number is less sensitive)

Go through different channels in the “Microscope Configuration” (see figure from step 7) that will be used in the experiment to adjust exposure time, camera analog gain and illumination intensity for each channel.

• analog gain of 6 (dB) is recommended for most settings
• note that illumination intensity value is proportional to the current set point for the laser diode driver, which means illumination set at low value will result in no output (because set current is below threshold current). Over the next 2-3 months we’ll make available a module for automatically mapping the output power and set current so that output power can be set directly.
• if after setting exposure time the live stream stops updating, disable live and enable live again

Configure laser autofocus. With the sample is in focus, turn off Live and click the “Initialize” button in the “Laser Autofocus Control” tab:

• “Initialize” will (1) locate the laser spot and set ROI to increase speed and (2) calculate the pixel to um conversion by measuring the location of the laser spot on the sensor at two z positions. If successful, “Laser-Based Focus” tab will display the following with one or two spots depending on objective selection.

  

• After Initialization, it’s recommended to turn on Live, make sure the sample is in focus, turn off live, and then click “Set as reference plane” button in the “Laser Autofocus Control” tab

Doing Multi-channel acquisition. To take an image or the current FOV or a grid of image around the current FOV, use the Flexible Multipoint tab

• step 1: choose where the output should be saved (default is Downloads)
• step 2: enter identifying information in the ID entry. This will be the name of the output folder that will be created. Date and time will be appended automatically
• step 3: configure the scan
  • fields
    • Nx is the number of FOV along the x direction
    • Ny is the number of FOV along the x direction
    • Nz is the number of z stacks
    • Nt is the number of time points
    • dx (mm) is the spacing between the FOV along the x direction
    • dy (mm) is the spacing between the FOV along the y direction
    • dz (um) is the spacing between z planes
  • setting dx (mm) and dy (mm)
    • For setup with regular tube lens and IMX571 sensor, pixel size is 0.752 um at 10x with 2 x 2 binning and the FN26.5 objective rated FOV on the sensor is around 1.567 mm along y direction and 2.142 mm along x direction (at 10x). The full sensor frame FOV is 1.56 mm x 2.348 mm.
    • For setup with f = 50 mm tube lens and IMX226 sensor, pixel size is 0.667 um at 10x and the FN26.5 objective rated FOV is 1.873 mm x 1.873 mm (at 10x).
  • if Nx > 1 and/or Ny > 1, the resulting FOV will be centered around the current location (or the locations in the location list)
• step 4: select the channels that will be used. selected channels will be highlighted in blue.
• step 5: check the “Reflection AF” checkbox if you want to use laser autofocus (will be performed at every FOV)
• step 6: click “Start Acquisition”

Doing plate scan. For imaging certain one or multiples of a plate, select the wells to be imaged in the Well Selector widget (control + click or drag across the wells) and follow the same instructions above (except that “Multipoint (Wellplate)” tab should be used

Load another sample. Make sure the microscope is not in the “Live” mode so that illumination is off. One can switch the key to the OFF location on the laser engine as extra caution.

Turn off the microscope after experiments.

1. close the software.
2. switch off the laser engine using the master switch on the back of the laser engine or the switch on the power strip.
3. optionally turn off the power to the microscope controller and the camera using the switch(es) on the power strip.