Phasor approach#

“The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling and storage. The reciprocity principle between the real and imaginary space—where the phasor and the pixel that the phasor originated from are linked and can be converted from one another—has helped the expansion of this method. The phasor coordinates calculated from a pixel, where multiple fluorescent species are present, depends on the phasor positions of those components. The relative positions are governed by the linear combination properties of the phasor space. According to this principle, the phasor position of a pixel with multiple components lies inside the polygon whose vertices are occupied by the phasor positions of these individual components and the distance between the image phasor to any of the vertices is inversely proportional to the fractional intensity contribution of that component to the total fluorescence from that image pixel. The higher the fractional intensity contribution of a vertex, the closer is the resultant phasor. The linear additivity in the phasor space can be exploited to obtain the fractional intensity contribution from multiple species and quantify their contribution.” (quoted from Malacrida et al., 2021)

The following resources provide an overview of the history, theory, applications, and implementations of the phasor approach:

Wiki#

Talks#

Articles#

Software#

Besides the PhasorPy library, several other software implemented the phasor approach to analyze fluorescence time-resolved or spectral images:

  • Globals for Images · SimFCS is a free, closed-source, Windows desktop application for fluorescence image analysis, visualization, simulation, and acquisition. The software was developed by Enrico Gratton during 1998-2022 at the Laboratory for Fluorescence Dynamics. It provides the most comprehensive set of features for phasor analysis of fluorescence lifetime and hyperspectral images. Many tutorials are available.

  • Spectral Phasor PlugIn and Time Gated Phasor PlugIn are open-source ImageJ plugins by Farzad Fereidouni, which provide visualization, segmentation, and unmixing of time-resolved and spectral images using the phasor approach. The software is distributed under an unknown license and was last updated in 2013.

  • Napari-flim-phasor-plotter is a napari plugin to interactively load and show raw FLIM single images and series and generate phasor plots.

  • Napari-live-flim is a napari plugin for real-time TCSPC-FLIM analysis based on FLIMLib. The plugin is distributed under the GPLv3 license.

  • HySP is a free, closed-source, multi-platform desktop application developed by Francesco Cutrale for the analysis of multi-dimensional, hyper- and multi-spectral data using the phasor approach.

  • FLUTE, the Fluorescence Lifetime Ultimate Explorer, is an open-source Python GUI for interactive phasor analysis of FLIM data developed by Chiara Stringari and others. The software is distributed under the GPL license.

  • FLIM_tools is an open-source Python library for linear unmixing and phasor tools for FLIM analysis developed by Jay Unruh. The library is distributed under the GPL2 license.

  • tttrlib is an open-source C++ and Python library to read, write, and process time-tagged time-resolved (TTTR) data. It includes functions for computing and correcting phasor coordinates. The library is distributed under the BSD license.

  • PhasorIdentifier is a Jupyter notebook to analyze FLIM files, including masking, cell segmentation, pH correlation, nanoscale effects, and precise quantification. The notebook is distributed under CC BY-NC 4.0.

  • FLIMLib is an exponential curve fitting library used for Fluorescent Lifetime Imaging (FLIM). It includes a function to calculate phasor coordinates from time-resolved signals. FLIMLib is licensed under the GPL v3.

  • FLIMfit is an open-source, MATLAB-based tool to analyse and visualize time-resolved data from FLIM measurements including TCSPC and wide-field time-gated imaging. It includes a segmentation tool based on the phasor approach. The affiliated FlimReader library provides file readers for FLIM data including TTTR. FLIMfit is licensed under the GPL v2.

  • PAM is an open-source GUI-based MATLAB package for the analysis of fluorescence experiments. The phasor analysis includes region of interests, fractions, triangular mixtures, and FRET estimation. PAM is licensed under the GPL v3.

  • Instant-FLIM-Analysis is an open-source MATLAB-based program that analyzes data acquired with an “instant FLIM” system. It supports image segmentation based on phasor plot regions of interest and K-means clustering.

  • FLIM Studio is a commercial software by FLIM LABS, a vendor of portable devices for fluorescence lifetime imaging and spectroscopy. The software provides real-time FLIM phasor-plot analysis, AI-driven phasor-plot analysis techniques, and an application programming interface. Python modules by FLIM LABS are available on GitHub.

  • VistaVision is a commercial Windows desktop software by ISS, Inc., for confocal microscopy applications, including instrument control, data acquisition, and data processing. It performs image segmentation of FLIM images via the phasor plot.

  • SPCImage is a commercial Windows desktop software by Becker & Hickl (tm) for TCSPC-FLIM data analysis. It performs image segmentation of time-resolved data via the phasor plot.

  • LAS X is a commercial Windows desktop software by Leica Microsystems. The software allows, by using phasors, to follow microenvironmental changes, select components to multiplex signal, and determine FRET efficiency.

  • Luminosa is a commercial Windows desktop software by PicoQuant GmbH, which includes single molecule detection, FCS, and time-resolved imaging methods. The InstaFLIM module allows simultaneous TCSPC and phasor analysis options for ROI determination.

  • EzTime is a commercial software by HORIBA Scientific. It allows real-time TCSPC image analysis and visualization, including phasor plots, in conjunction with the FLIMera wide-field camera.

  • FlimFast was a research-grade Windows desktop software for frequency-domain, full-field, fluorescence lifetime imaging at video rate, developed by Christoph Gohlke during 2000-2002 at UIUC. It enabled phasor vs intensity plots of FLIM images during real-time acquisition.

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