Fig. 1
From: Spectral phasor imaging on a commercial confocal microscope without a spectral detector
Spectral phasor using 4 channels of a commercial laser scanning microscope. (a–d) Schematic of the 4 C-spectral phasor method: (a) 4-color images are acquired using a commercial laser scanning microscope; (b) Emission spectra variations are sampled across four spectral detection windows; (c) For each pixel, a phasor in the (g, s) plane is computed, where phasor position shifts reflect pixel-to-pixel emission spectra variations; (d) a phase wavelength image (\(\:{{\uplambda\:}}_{\text{φ}}\)) is calculated converting the value of phase into a value of wavelength. (e–g) Simulated data used for reference line generation. The spectral bandwidth of each detection channel is set to 50 nm. (e, top) Simulated Gaussian spectra with a narrow bandwidth (FWHM = 10 nm) and peak wavelength increasing from the value λ1 (indicated by the arrow) in steps of 5 nm. (e, middle) Phasor plot of the simulated spectra. The red values (λ1, λ2, λ3, λ4) represent the center wavelengths of the 4 detection windows. Each red dot corresponds to a single spectrum. The narrow spectra are not uniformly sampled by the spectral windows, producing the polygonal trajectory. (e, bottom) Plot showing the phase value of the phasor versus the simulated peak wavelength for the 4C- and for the full spectral phasor. (f, top) Simulated Gaussian spectra with a broader bandwidth (FWHM = 100 nm) and peak wavelength increasing from the value λ1 (indicated by the arrow) in steps of 5 nm. (f, middle) Phasor plot of the simulated spectra. Each red dot corresponds to a single spectrum. The broad spectra are more uniformly sampled, resulting in a curved trajectory, where each dot represents a 5 nm shift in peak wavelength. (f, bottom) Plot showing the phase value of the phasor versus the simulated peak wavelength for the 4C- and for the full spectral phasor. (g, top) Simulated Gaussian spectra of fixed center wavelengths (λ1, λ2, λ3, λ4) and bandwidths increasing from 0 to the value 400 nm in steps of 20 nm. (g, middle) Phasor plot of the simulated spectra. Each red dot corresponds to a single spectrum. These simulated spectra produce radial-like phasor trajectories.
