Illumination Power, Stability, and Linearity Measurements for Confocal, Widefield and Multiphoton Microscopes v1 Article Swipe

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Nathalie Gaudreault , Luis-Francisco Acevedo-Hueso , Rizwan Ali , Sergiy Avilov , Steve Bagley , Rodrigo Roberto Bammann , Fábio Barachati , L Barna , Valeria Berno , Cristina Bertocchi , Veronika Boczonadi , Ulrike Boehm , Manel Bosch , Craig Brideau , Mariana T. Carvalho , N. Chatterjee , Pina Colarusso , Richard Cole , N. Darwish , Suat Dervish , Ralf Dietzel , Sam Duwé , Frank Eismann , Orestis Faklaris , Andreas Felscher , Zannatul Ferdous , Thomas Guilbert , Manfred Gonnert , David Grünwald , Tingting Gu , Birgit Hoffmann , Marcel Kirchner , Ayse Aslihan Koksoy , Gabriel Krens , Alex Laude , Jeffrey LeDue , Penghuan Liu , Pascal Lorentz , Gabriel G. Martins , Valeria.MezzanoRobinson not provided , Mišo Mitkovski , Glyn Nelson , Michael S. Nelson , Roland Nitschke , Alex L. Payne-Dwyer , Jaime A. Pimentel , Santosh Podder , Britta Schroth‐Diez , Stanley Schwartz , Ronian Siew , Sathya Srinivasan , Roland Thuenauer , Kees van der Oord , Douglas J. Taatjes , Chloë van Oostende‐Triplet , Tse-Luen Wee , Kurt Weiss , Laurent Gelman ·

YOU? · · 2024 · Open Access · · DOI: https://doi.org/10.17504/protocols.io.n2bvj3m45lk5/v1

To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on different microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and may, therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time or even between two microscopes of the same model. In addition to illumination stability, working within the linear range of the illumination power allows to adjust accurately the illumination power absolute value (in mW) using a fraction (or %) of its maximal value through the imaging software. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is intended for confocal systems (raster scanning and spinning disks), Multi-photon systems (used for 2P- or 3P-imaging, SHG-imaging, etc.), and widefield systems. It represents the collective experience of over 50 imaging scientists.

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Confocal Microscope Optics Linearity Stability (learning theory) Confocal microscopy Power (physics) Materials science Physics Computer science Quantum mechanics Machine learning

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Type: preprint
Language: en
Landing Page: https://doi.org/10.17504/protocols.io.n2bvj3m45lk5/v1
OA Status: gold
Related Works: 10
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DOI: https://doi.org/10.17504/protocols.io.n2bvj3m45lk5/v1

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Title: Illumination Power, Stability, and Linearity Measurements for Confocal, Widefield and Multiphoton Microscopes v1

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Language: en

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Publication year: 2024

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Publication date: 2024-01-19

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Authors: Nathalie Gaudreault, Luis-Francisco Acevedo-Hueso, Rizwan Ali, Sergiy Avilov, Steve Bagley, Rodrigo Roberto Bammann, Fábio Barachati, L Barna, Valeria Berno, Cristina Bertocchi, Veronika Boczonadi, Ulrike Boehm, Manel Bosch, Craig Brideau, Mariana T. Carvalho, N. Chatterjee, Pina Colarusso, Richard Cole, N. Darwish, Suat Dervish, Ralf Dietzel, Sam Duwé, Frank Eismann, Orestis Faklaris, Andreas Felscher, Zannatul Ferdous, Thomas Guilbert, Manfred Gonnert, David Grünwald, Tingting Gu, Birgit Hoffmann, Marcel Kirchner, Ayse Aslihan Koksoy, Gabriel Krens, Alex Laude, Jeffrey LeDue, Penghuan Liu, Pascal Lorentz, Gabriel G. Martins, Valeria.MezzanoRobinson not provided, Mišo Mitkovski, Glyn Nelson, Michael S. Nelson, Roland Nitschke, Alex L. Payne-Dwyer, Jaime A. Pimentel, Santosh Podder, Britta Schroth‐Diez, Stanley Schwartz, Ronian Siew, Sathya Srinivasan, Roland Thuenauer, Kees van der Oord, Douglas J. Taatjes, Chloë van Oostende‐Triplet, Tse-Luen Wee, Kurt Weiss, Laurent Gelman

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Landing page: https://doi.org/10.17504/protocols.io.n2bvj3m45lk5/v1

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Concepts: Confocal, Microscope, Optics, Linearity, Stability (learning theory), Confocal microscopy, Power (physics), Materials science, Physics, Computer science, Quantum mechanics, Machine learning

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