Hyperspectral Analysis of Fluorescence Images in Scattering Media using Optical Filters

Abstract

We present the simple fluorescence imaging technique that allows hyperspectral analysis of images even obtained in highly scattering media. This technique can be applied to any fluorescence microscope equipped with a single detector or camera. The conventional hyperspectral imaging usually utilizes spectrometers embedded in a microscope, which are designed for use with collimated light and thus not efficient for imaging in scattering samples, such as tissues. Also, this is an expensive addition to the microscope. In the proposed method, we utilize optical filters which have certain transmittance characteristics in the spectral range of interest. The images are collected through these filters, either in sequence or parallel, and then the spectral phasor coordinates can be calculated to perform hyperspectral analysis. Several types of filters can be used in this method, including filters with sin-cos and linear transmittance profiles. The sin-cos filters allow recovering information on central wavelength and width of fluorophore emission spectra, and the obtained spectral phasors follow the rule of linear addition. Use of linear filters simplifies the measurements, because only a single filter is needed, but it recovers only the central wavelength with great resolution. Using this linear filter we can achieve accuracy of few nm to separate various fluorophores, which can be sufficient for applications in which there is spatial separation of the dye or of the spectra of the dyes. We have applied this method to dyes solutions and imaging several biological samples, such as cells and tissues with different dyes. These results were comparable with ones obtained on the commercial Zeiss-LSM710 microscope. Several commercial low-cost plastic filters were used in this method; we have also fabricated filters with better characteristics using a mixture of dyes dispersed in a polymer host (Supported by NIH grantP41-M103540).