Microscopy Data Analysis Using Phasor Plot Histograms
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Solution Overview
Problem
Existing methods for analyzing microscopy data, particularly light microscopy data, face challenges in determining the relative contribution of different emitter species to detected light, especially when dealing with mixed emission spectra or lifetimes, which complicates spectral unmixing and lifetime component separation.
Innovation Solution
A method involving the use of a processor to receive and analyze microscopy data, determining the relative contribution of different emitter species to detected light by displaying the data in a histogram representing relative contributions to channels, and allowing user input to select and adjust the data subsets based on the histogram.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If time-gated detection is used to separate lifetime components, then lifetime components can be separated, but the signal-to-noise ratio is reduced
Solution Approach 1:
The patent transforms the time-domain fluorescence decay data into the frequency domain using Fourier transformation. By analyzing the real and imaginary parts of the Fourier coefficients in a phasor plot, the method separates lifetime components without discarding photons, thus maintaining signal-to-noise ratio while achieving accurate lifetime component separation.
2Measurement precision
If hardware-implemented binning is used for lifetime unmixing, then lifetime component distribution can be obtained, but dedicated hardware is required and time information is lost
Solution Approach 1:
The patent replaces the mechanical hardware binning system with a computational approach. By performing Fourier transformation on the photon arrival time data and analyzing the resulting phasor plot, the method achieves lifetime unmixing through software processing rather than hardware binning, eliminating the need for dedicated hardware while preserving time information.
3Measurement precision
If spectral unmixing is performed to determine relative contribution of emitter species, then channel separation is achieved, but the process is complicated especially when reference spectra are not available
Solution Approach 1:
The patent replaces complex spectral unmixing algorithms with phasor analysis. By transforming fluorescence spectra into the frequency domain and representing them as vectors in a phasor plot, the method simplifies the determination of relative contributions of different emitter species. The phasor approach provides an intuitive geometric interpretation that eliminates the need for reference spectra and complex iterative unmixing procedures.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach provides an intuitive and comprehensive tool for analyzing microscopy data, allowing users to easily select and allocate data to channels, even for inexperienced users, thereby improving the accuracy and efficiency of determining relative contributions of emitter species.
Implementation Method 1
different fluorophores, which have a distinct fluorescence emission spectrum or fluorescence lifetime
Data Source
Figure 1A~2
Figure 3A~3F
Figure 4
AI summary
The invention relates to a method, a device (100) and a computer program for analyzing microscopy data (1), comprising the steps of providing microscopy data obtained from a sample (2) comprising a plurality of emitters (3), wherein the microscopy data (1) comprise emittance light intensities for a plurality of pixels (4), determining for each of the pixels (4) a relative contribution to a plurality of channels (A, B, C) representing emittance light emitted by different species of the emitters (3), displaying the microscopy data (1) in at least one histogram (10) comprising a first axis (11) representing the relative contributions to the channels (A, B, C), displaying a manipulation tool (20), receiving a user input indicating a manipulation of the manipulation tool (20) to select a subset of the microscopy data (1) from the histogram (10) or to adjust the relative contribution to the channels (A, B, C), and displaying at least one image(31,32) based on the selected subset of the microscopy data (1) and/or the adjusted relative contributions to the channels (A, B, C).