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Segmentation-guided photon pooling enables robust single cell analysis and fast fluorescence lifetime imaging

Kayvan Samimi1, Danielle E Desa1, Xiaotian Zhang2

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This study introduces a photon pooling method to speed up fluorescence lifetime imaging microscopy (FLIM) for analyzing living cells. This technique enables faster, label-free metabolic analysis of single cells and large imaging areas.

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Area of Science:

  • Cellular and Molecular Imaging
  • Biophotonics
  • Metabolic Imaging

Background:

  • Fluorescence lifetime imaging microscopy (FLIM) offers label-free, non-invasive probing of cellular metabolic states.
  • Current FLIM methods require long acquisition times due to low signal from endogenous fluorophores, hindering real-time analysis.
  • Accurate metabolic profiling necessitates high photon counts for precise multi-exponential decay fitting at the pixel level.

Purpose of the Study:

  • To develop a faster method for label-free, single-cell FLIM acquisition and analysis.
  • To overcome the limitations of long integration times in conventional FLIM.
  • To enable rapid, high-resolution metabolic imaging of living cells.

Main Methods:

  • Implementation of a 'region-of-interest' photon pooling technique.
  • Acquisition and analysis of FLIM data using the novel photon pooling approach.
  • Comparison of acquisition speed and data quality against conventional pixel-level analysis.

Main Results:

  • Achieved single-cell metabolic information at acquisition intervals as short as one second.
  • Enabled the acquisition of large FLIM mosaics 15 times faster than conventional methods.
  • The technique is computationally efficient, requires no machine learning, and integrates with standard software.

Conclusions:

  • The 'region-of-interest' photon pooling technique significantly accelerates label-free FLIM.
  • This method provides a practical solution for rapid, high-throughput cellular metabolic analysis.
  • The developed technique enhances the utility of FLIM for studying dynamic cellular processes.