Place a lens tissue on the sample stage above the objective lens to serve as a diffuser.

Acquire brightfield mode images from the microscope with a 100ms exposure time, capturing at least 500 frames consecutively under no light. Ensure EM Gain is off.

Repeat step 2 with increasing exposure times (at least 5 times, including step 2).

Save the images taken under different exposure times in the 'camera_calibration' folder in .tif format and prefix the file names with 'exposure_'. For instance, the first image captured under no light should be named '0_filename', and the second image taken with slightly increasing exposure should be named '1_filename'.

Acquire brightfield mode images from the microscope with a 100ms exposure time, capturing at least 500 frames consecutively under no light. Ensure EM Gain is set to the setting of interest ( e.g. 250).

Repeat step 2 with increasing exposure times (at least 5 times, including step 2). Ensure no pixels are above/at saturation.

Save the images taken under different exposure times in the 'camera_calibration_EMGain_N' folder in .tif format and prefix the file names with 'exposure_'. For instance, the first image captured under no light should be named '0_filename', and the second image taken with slightly increasing exposure should be named '1_filename'.

Use the GDSC-SMLM plugin for imageJ to calculate gain at no gain, ("Mean-Variance Test") to calculate camera gain.Then, using this gain parameter, use the "Mean-Variance Test (EM-CCD)" function to calculate the overall game and offset.