Abstract
A new global analysis algorithm to analyse (hyper-) spectral images is presented. It is based on the phasor representation that has been demonstrated to be very powerful for the analysis of lifetime imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the image is Fourier transformed. Next, the real and imaginary components of the first harmonic of the transform are employed as X and Y coordinates in a scatter (spectral phasor) plot. Importantly, the spectral phasor representation allows for rapid (real time) semi-blind spectral unmixing of up to three components in the image. This is demonstrated on slides with fixed cells containing three fluorescent labels. In addition the method is used to analyse autofluorescence of cells in a fresh grass blade. It is shown that the spectral phasor approach is compatible with spectral imaging data recorded with a low number of spectral channels.
Highlights
Fluorescence microscopy is commonly used to studylocalization of multiple components in biological specimen [1]
The colour of the pixels in the phasor plot corresponds to the colour of fluorescence emission in that pixel as perceived by eye
For reference purposes a semicircle of theoretical Gaussian emission spectra with maxima ranging from λem = 370 to 650 nm and width ∆λ = 250 cm−1 is shown in the phasor plot
Summary
Fluorescence microscopy is commonly used to study (co)localization of multiple components in biological specimen [1]. These components can be distinguished by their spectroscopic properties, and simultaneously imaged by for instance (hyper) spectral imaging (SI) [2, 3], fluorescence lifetime imaging (FLIM) [4,5,6,7], or combinations of these techniques. The reference spectra are usually based on literature data or obtained in separate reference measurements The use of such reference spectra can introduce artefacts because of changes in the spectra caused by solvatochromic shifts or other environmental effects. To correct for these effects, extensive calibration efforts are required
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