Britta Schroth-Diez, Kees van der Oord, Ruediger Bader1. Light Microscopy Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden Germany
2. Nikon Europe BV
3. Hamamatsu Photonics Europe
QUAREP-LiMi Working Group 2 (WG2) developed a set of protocols [1] which focuses on the characterization and monitoring of the performance of light microscope detection systems, which collects light from the sample at the microscope and helps to convert the “arbitrary digital units” (ADU) provided by the microscope into “number of photons” detected.
The protocols collection is built around the photon transfer curve (PTC) method [2]. The goals of these protocols are to achieve 1) experiment quality control, 2) monitoring instrument quality over time, and 3) detailed detection system characterization. The protocols include both point and area detectors, allowing researchers to select appropriate methods based on their specific detection systems.
During the workshop, we will demonstrate how to use the protocols to acquire images and upload them in the software [3] to characterize the photon conversation factor (PCF photons/ADU), readnoise, background and dynamic range of a light microscopy detection system. The set includes protocols on sample preparation, data acquisition, and analysis. Furthermore, we will demonstrate its integration in the QUAREP-LiMi Tool Kit [4].
[1] Characterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems – https://dx.doi.org/10.17504/protocols.io.14egn61pyl5d/v1
[2] Janesick JR. 2007. Photon Transfer. SPIE. https://dx.doi.org/10.1117/3.725073
[3] McFadden D. 2022. GUI Calibration Tool. https://github.com/bionanoimaging/NanoImagingPack/releases
[4] QUAREP-LiMi Tool Kit, https://github.com/QUAREP-LiMi/QUAREP-LiMi-Tool-Kit