Abstract
Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that 2 filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than displaying the entire spectrum. Using the properties of the spectral phasors, we can resolve spectral components and perform the type of data analyses that are usually performed in hyperspectral image analysis.
Highlights
Spectral phasors were recently introduced as an alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation of probes in membranes [1,2,3,4,5,6,7,8]
The basic concept in the spectral phasor approach is that the entire spectrum is not needed for some of the classical spectral analysis techniques such as demixing, but only few parameters of the spectral distribution are sufficient for these calculations
We introduced in 2011 a microscope design that can capture a very large solid angle of the emitted light and we have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes [13]
Summary
Spectral phasors were recently introduced as an alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation of probes in membranes [1,2,3,4,5,6,7,8]. In the case in which the spectrum is dominated by few spectral bands, this approach is very efficient and provides fast and valid alternatives to the analysis of the full spectrum. This is convenient in cases like spectral analysis during surgery, in live animals studies [9,10] and in all those cases in which full hyperspectral imaging is not feasible or inconvenient. One example of a system that could greatly benefit from the phasor analysis approach in tissue spectroscopy (absorption and fluorescence) is the case in which the light to be collected has undergone multiple scattering. We show here that plastic filters that can be used for this purpose are available (Cyan and Green filters from Neewer 18x20cm Transparent Color Correction Lighting Gel Filter Set, available for $10.99 on Amazon cat# B016Q0BA6A) and we describe their use in the DIVER microscope to obtain hyperspectral information using the spectral phasor approach
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