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Spectral phasor imaging on a commercial confocal microscope without a spectral detector
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Journal Article
Spectral phasor imaging on a commercial confocal microscope without a spectral detector
2025
Overview
Spectral imaging is a fluorescence microscopy technique with several applications, including imaging of environment-sensitive probes, spectral unmixing and identification of fluorescent species. In confocal microscopes not equipped with a spectral detection unit, spectral images can be obtained using the lambda scan mode of the microscope, namely the sequential acquisition of images using a tunable emission filter or other dispersive optical elements. Unfortunately, the lambda scan mode has poor temporal resolution, is a photon-wasting technique, and is not ideal for the spectral imaging of live samples. Here, we describe a spectral imaging method that can be implemented on commercial confocal microscopes not equipped with a spectral detector. The method is based on simultaneous image acquisition in 4 contiguous spectral channels and spectral phasor analysis. We demonstrate that this method can be easily implemented on a Leica confocal laser scanning microscope, with better photon efficiency and temporal resolution than the lambda scan mode. We perform a 4-channel (4 C) spectral phasor analysis of live cells stained with the environment-sensitive ACDAN and Nile Red dyes. We can distinguish changes in spectral emission in the order of 5 nm between different subcellular compartments. We show that 4 C-spectral phasor can be used to decompose the Nile Red signal into 2 components and perform 3-color imaging in combination with a DNA dye in live organoids. Finally, we show that the 4 C-spectral phasor can be also used to unmix the signal of fluorescent proteins with overlapping emission spectra such as mEmerald and EYFP.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ ACDAN
/ Biopsy
/ Colon
/ Dyes
/ Environment-sensitive probes
/ Fluorescent Dyes - chemistry
/ Humanities and Social Sciences
/ Labeling
/ Microscopy, Confocal - instrumentation
/ Microscopy, Confocal - methods
/ Microscopy, Fluorescence - methods
/ Nile red
/ Plasmids
/ Proteins
/ Science
/ Sensors
