Jan 13, 2025

Public workspaceCharacterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems

DOI
https://dx.doi.org/10.17504/protocols.io.14egn61pyl5d/v1
Characterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems
  • David McFadden1,
  • Laszlo Barna2,
  • Luis-Francisco Acevedo-Hueso3,
  • Sergiy V Avilov4,
  • Gert-Jan Bakker5,
  • Sebastian Beer6,
  • Ivan Belyaev7,
  • Valeria Berno8,
  • Mariana T Carvalho9,
  • Yann Cesbron10,
  • Catalin Chiritescu11,
  • Orestis Faklaris12,
  • Nathalie Gaudreault13,
  • David Grunwald14,
  • Thomas Guilbert15,
  • Mathias Hammer16,
  • Rainer Heintzmann17,
  • Gerhard Holst18,
  • Ayse Aslihan Koksoy19,
  • Gabriel Martins20,
  • Glyn Nelson21,
  • Roland Nitschke22,
  • Alex Payne-Dwyer23,
  • Santosh Podder24,
  • Sathya Srinivasan25,
  • Kees van der Oord26,
  • Andre Zeug27,
  • Britta Schroth-Diez28
  • 1Friedrich Schiller University Jena;
  • 2Institute of Experimental Medicine, Budapest;
  • 3Neuro-AI Monitoring and Quantum Medical Diagnostics;
  • 4Max Planck Institute of Immunobiology and Epigenetics;
  • 5Radboud University Medical Center, Medical BioSciences department, Nijmegen, the Netherlands;
  • 6Hamamatsu Photonics Deutschland GmbH;
  • 7The Microverse cluster, Friedrich Schiller University Jena;
  • 8ALEMBIC Advanced Light and Electron Microscopy BioImaging Center, San Raffaele Scientific Institute;
  • 9INL - International Iberian Nanotechnology Laboratory;
  • 10Institute of Science and Technology Austria;
  • 11Phi Optics;
  • 12MRI, Biocampus, University of Montpellier, CNRS, INSERM, Montpellier, France;
  • 13Allen Institute for Cell Science, Seattle, WA, USA;
  • 14UMass Chan Medical School;
  • 15Institut Cochin, INSERM U1016, CNRS UMR 8104, Université Paris Cité, Paris, France;
  • 16UMass Chan Medical School, RNA Therapeutics Institute, Worcester, MA, USA;
  • 17Leibniz Institute of Photonic Technology;
  • 18Excelitas PCO GmbH;
  • 19MD Anderson Cancer Center;
  • 20Gulbenkian Institute for Molecular Medicine;
  • 21Newcastle University, BioImaging Unit, UK;
  • 22Life Imaging Center, University of Freiburg, Germany;
  • 23School of Physics Engineering and Technology, University of York, UK;
  • 24Indian Institute of Science Education and Research (IISER) Pune;
  • 25ONPRC;
  • 26Nikon Europe BV;
  • 27Cellular Neurophysiology, Hannover Medical School, Hannover, Germany;
  • 28Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-Diez
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn61pyl5d/v1
External link: https://quarep.org/
Collection Citation: David McFadden, Laszlo Barna, Luis-Francisco Acevedo-Hueso, Sergiy V Avilov, Gert-Jan Bakker, Sebastian Beer, Ivan Belyaev, Valeria Berno, Mariana T Carvalho, Yann Cesbron, Catalin Chiritescu, Orestis Faklaris, Nathalie Gaudreault, David Grunwald, Thomas Guilbert, Mathias Hammer, Rainer Heintzmann, Gerhard Holst, Ayse Aslihan Koksoy, Gabriel Martins, Glyn Nelson, Roland Nitschke, Alex Payne-Dwyer, Santosh Podder, Sathya Srinivasan, Kees van der Oord, Andre Zeug, Britta Schroth-Diez 2025. Characterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn61pyl5d/v1
License: This is an open access collection 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 collection and it's working
Created: September 08, 2024
Last Modified: September 01, 2025
Collection Integer ID: 107127
Keywords: protocol collection, detection system, characterization, sample preparation, in-homogeneous, photon conversion factor, dark noise, dynamic range, light microscope, photon transfer method, quality control, reproducibility, light microscopy detection system, light microscope detection system, monitoring of microscope performance, dynamic range of light microscope detection system, microscope performance, photon conversation factor, detector, area detector, point detector, detection setting, detection system, experiment qc, imaging scientist, homogeneous detector illumination scheme, characterization of the photon conversion factor, using detection setting, suitable sample for the acquisition, photon conversion factor, detection, pcf photo, uniform illumination, monitoring, full characterization of detection path performance, respective analysis protocol, instrument qc, detection path performance, protocol collection, image quality, protocols in this collection, aims this protocol, experiment, Quarep
Disclaimer
This protocol collection was developed by members of WG2 "Detection System Performance" of the international consortium QUAREP-LiMi.
The Consortium for Quality Assessment and Reproducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi), formed by the global community of practitioners, researchers, developers, service providers, funders, publishers, policy makers and industry related to the use of light microscopy, is committed to democratizing access to quantitative and reproducible light microscopy and the data generated by it.
This protocol collection has undergone the internal approval process of QUAREP-LiMi.
It is not the intention of this protocol collection to supplant the need for independent professional judgement, advice, diagnosis or treatment. Any action taken or withheld on the basis of the information presented here is undertaken at the user's own risk. The user agrees that neither the company nor any of the authors, contributors, administrators, or other individuals associated with protocols.io or QUAREP-LiMi can be held liable for any injuries, damages or losses incurred as a result of the user's use of the information contained in or linked to this protocol or any of our websites, applications, or services.
Abstract
The protocols in this collection describe how to measure and analyze the photon conversation factor (PCF photo-electrons/count), readnoise, and dynamic range of a light microscopy detection system; which can either be a point detector or an area detector, using an in-homogeneous detector illumination scheme (as opposed to uniform illumination). The collection includes protocols on how to prepare a suitable sample for the acquisition, how to acquire data, as well as a respective analysis protocol.

There are three aims a detection system can be characterized for, briefly:
Aim 1 - experiment QC: Characterize the microscope performance using detection settings that match the experiment.
Aim 2 - instrument QC.: Monitoring of microscope performance over time for service purposes, to maintain image quality constant and at high level.
Aim 3 - system characterization: Full characterization of detection path performance under the range of settings applied by users.

For a more detailed description refer to protocol 1. Introduction - Background and Aims.
Workflow of detection system characterization using the Light Microscopy Detection System Characterization toolset.
This protocol collection represents the collective experience of over 100 imaging scientists and industry experts. Measurements made by our working group with these protocols will be available in a public database.

Please note, that this is an evolving document, to be versioned and updated, based on community feedback and new data.
Materials
For materials please refer to the repsective protocol.
Troubleshooting
Safety warnings
Ensure you follow general lab safety guidelines for radiation sources and chemicals as outlined within your organisation.



Laser safety and regulations
  • Please refer to the documentation provided by the manufacturer for additional warnings and preventive, protective equipment (PPE) requirements (e.g. laser safety goggles). Always consult your local Laser Safety Officer or Radiation Safety Officer and refer to your laboratory safety documentation for more information.
  • You can also consult your Laser Safety Standards ANSI Z136 in North America, SUVA 66049.D in Europe, and BS EN 60825-1 in the UK. Additionally, laser safety standards and regulations are covered by IEC norm 60825-1, and LED eye safety standards and regulations are covered by IEC norm 62471 in Europe.
  • Safety guideline: Hazardous, visible, or invisible radiation from lasers, lamps, and other light sources used for microscopy can cause permanent damage to the retina, skin burns, and fire. Always follow proper laser safety protocols for your equipment and situation.
Before start
For a detailed introduction and description, please read protocol 1.
For characterization of an area detector (e.g., sCMOS camera), please follow protocol 2 , protocol 3, and protocol 5.
For characterization of a point detector (e.g., PMT or HyD detector), please follow protocol 2, protocol 4 and protocol 5.

Make sure the microscope is turned on at least one hour prior to the measurements to allow the system to stabilize.
The test sample can be prepared well in advance.

This protocol collection was developed by members of WG2 "Detection System Performance" of the international consortium QUAREP-LiMi.
The Consortium for Quality Assessment and Reproducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi), formed by the global community of practitioners, researchers, developers, service providers, funders, publishers, policy makers and industry related to the use of light microscopy, is committed to democratizing access to quantitative and reproducible light microscopy and the data generated by it.
This protocol collection has undergone the internal approval process of QUAREP-LiMi.
It is not the intention of this protocol collection to supplant the need for independent professional judgement, advice, diagnosis or treatment. Any action taken or withheld on the basis of the information presented here is undertaken at the user's own risk. The user agrees that neither the company nor any of the authors, contributors, administrators, or other individuals associated with protocols.io or QUAREP-LiMi can be held liable for any injuries, damages or losses incurred as a result of the user's use of the information contained in or linked to this protocol or any of our websites, applications, or services.
Files
Protocol
Icon representing the file 1. Introduction - Background and Aims
Name
1. Introduction - Background and Aims
Version 1
, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-DiezMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Protocol
Icon representing the file 2. Sample Preparation - An Easy-to-Prepare Sample Slide
Name
2. Sample Preparation - An Easy-to-Prepare Sample Slide
Version 1
, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-DiezMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Protocol
Icon representing the file 3. Data Generation - Systems with an Area Detector
Name
3. Data Generation - Systems with an Area Detector
Version 1
, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-DiezMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Protocol
Icon representing the file 4. Data Generation - Systems with a Point Detector
Name
4. Data Generation - Systems with a Point Detector
Version 1
, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-DiezMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Protocol
Icon representing the file 5. Analysis - Characterization of the Photon Conversion Factor, Noise, and Dynamic Range
Name
5. Analysis - Characterization of the Photon Conversion Factor, Noise, and Dynamic Range
Version 1
, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Britta Schroth-DiezMax Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Protocol references
Art J. 1995. Photon detectors for confocal microscopy. In: Pawley J, editor. Handbook of Biological Confocal Microscopy. New York, NY: Springer. pp. 183–195. Cho EH, Lockett SJ. 2006. Calibration and standardization of the emission light path of confocal microscopes. Journal of Microscopy 223:15–25. doi:10.1111/j.1365-2818.2006.01598.x Deagle RC, Wee T-L (Erika), Brown CM. 2017. Reproducibility in light microscopy: Maintenance, standards and SOPs. The International Journal of Biochemistry & Cell Biology 89:120–124. doi:10.1016/j.biocel.2017.06.008 EMVA 1288. n.d. . European Machine Vision Association. https://www.emva.org/standards-technology/emva-1288/ Heintzmann R, Relich PK, Nieuwenhuizen RPJ, Lidke KA, Rieger B. 2016. Calibrating photon counts from a single image. doi:10.48550/ARXIV.1611.05654 Janesick JR. 2007. Photon Transfer. SPIE. doi:10.1117/3.725073 McFadden D. 2022. gui calibration tool. Releases · bionanoimaging, NanoImagingPack. https://github.com/bionanoimaging/NanoImagingPack/releases
McFadden D, Amos B, Heintzmann R. 2022. Quality control of image sensors using gaseous tritium light sources. Phil Trans R Soc A 380:20210130. doi:10.1098/rsta.2021.0130 Mullikin JC, Van Vliet LJ, Netten H, Boddeke FR, Van Der Feltz G, Young IT. 1994. Methods for CCD camera characterization In: Titus HC, Waks A, editors. Presented at the IS&T/SPIE 1994 International Symposium on Electronic Imaging: Science and Technology. San Jose, CA. pp. 73–84. doi:10.1117/12.175165 Murray JM. 2013. Practical Aspects of Quantitative Confocal MicroscopyMethods in Cell Biology. Elsevier. pp. 427–440. doi:10.1016/B978-0-12-407761-4.00018-X

Olevsko I, Szederkenyi K, Corridon J, Au A, Delhomme B, Bastien T, Fernandes J, Yip C, Oheim M, Salomon A. 2021. A simple, inexpensive and multi‐scale 3‐D fluorescent test sample for optical sectioning microscopies. Microscopy Res & Technique 84:2625–2635. doi:10.1002/jemt.23813 Plakhotnik T, Chennu A, Zvyagin AV. 2006. Statistics of single-electron signals in electron-multiplying charge-coupled devices. IEEE Trans Electron Devices 53:618–622. doi:10.1109/TED.2006.870572 QUAREP-LiMi. n.d. Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy. https://quarep.org/ Ryan DP, Dunlap MK, Gelfand MP, Werner JH, Van Orden AK, Goodwin PM. 2021. A gain series method for accurate EMCCD calibration. Sci Rep 11:18348. doi:10.1038/s41598-021-97759-6 van Vliet L, Sudar D, Young I. 1998. Digital fluorescence imaging using cooled charge-coupled device array cameras In: Celis JE, editor. Cell Biology: A Laboratory Handbook. Vol. 3. New york: Acad. Press. pp. 109–120.
Acknowledgements
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