Feb 01, 2024
EMCCD Gain and Offset Calibration
- 1Yusuf Hamied Department of Chemistry, University of Cambridge
Protocol Citation: Joseph S Beckwith 2024. EMCCD Gain and Offset Calibration . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3dpdg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94556
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000509
Abstract
EMCCD Gain and Offset Calibration
EMGain 0 Camera gain and offset measurement protocol
EMGain 0 Camera gain and offset measurement protocol
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'.
EMGain on Camera gain and offset measurement protocol
EMGain on Camera gain and offset measurement protocol
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'.
Gain calculation
Gain calculation