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<p>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&#xa0;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&#xa0;nm between different subcellular compartments. We show that 4&#xa0;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&#xa0;C-spectral phasor can be also used to unmix the signal of fluorescent proteins with overlapping emission spectra such as mEmerald and EYFP.</p>

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Spectral phasor imaging on a commercial confocal microscope without a spectral detector

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月26日

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  • Elisa Longo,
  • Angelita Costantino,
  • Alessio Andreoni,
  • Paolo Bosco,
  • Anna P. Privitera,
  • Silvia Scalisi,
  • Leonel Malacrida,
  • Massimo Gulisano,
  • Luca Lanzanò

摘要

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.