Fluorescence to measure light intensity

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of waveleng...

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Bibliographic Details
Published in:Nature methods Vol. 20; no. 12; pp. 1930 - 1938
Main Authors: Lahlou, Aliénor, Tehrani, Hessam Sepasi, Coghill, Ian, Shpinov, Yuriy, Mandal, Mrinal, Plamont, Marie-Aude, Aujard, Isabelle, Niu, Yuxi, Nedbal, Ladislav, Lazár, Dusan, Mahou, Pierre, Supatto, Willy, Beaurepaire, Emmanuel, Eisenmann, Isabelle, Desprat, Nicolas, Croquette, Vincent, Jeanneret, Raphaël, Le Saux, Thomas, Jullien, Ludovic
Format: Journal Article
Language:English
Published: New York Nature Publishing Group US 01.12.2023
Nature Publishing Group
Subjects:
631/1647/245/2225
631/92/96
Actinometry
Bioengineering
Bioinformatics
Biological Microscopy
Biological Physics
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Chemical Sciences
Dyes
Fluorescence
Fluorescent Dyes - chemistry
Imaging
Instrumentation and Detectors
Life Sciences
Light
Light intensity
Light sources
Luminous intensity
Measuring instruments
Microscopes
Microscopy, Fluorescence - methods
Optics
Physics
Proteins
Proteomics
Spatial distribution
Spectrometry, Fluorescence
Wavelengths
Chemical tools
Fluorescence imaging
ISSN:1548-7091, 1548-7105, 1548-7105
Online Access:Get full text
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Summary:Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources. Two methods for fluorescence-based actinometry using organic dyes and photoconvertible fluorescent proteins enable rapid and precise measurement of light intensity at the sample in fluorescence microscopes.
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ISSN:1548-7091
1548-7105
1548-7105
DOI:10.1038/s41592-023-02063-y