Implementation of a multiplexed structured illumination method to achieve snapshot multispectral imaging.

An instantaneous multispectral imaging setup based on frequency recognition algorithm for multiple exposures (FRAME) is presented and demonstrated experimentally. With this implementation of FRAME, each light source is uniquely encoded with a spatial modulation and the corresponding fluorescent responses pertaining to each maintain the same unique encoding. This allows the extraction of each source response from a single captured image by filtering in the Fourier domain. As a result, a multispectral imaging system based on FRAME can perform all the illumination and corresponding fluorescence detection simultaneously, where the latter is recorded in a single exposure and on a single detector and is thus capable of recording true 'snapshot' multispectral images. The results presented here demonstrate that the technique is capable of distinguishing source responses for well separated and co-localized fluorophores as well as providing z-sectioning capabilities. This implementation of FRAME demonstrates its viability as a tool for multispectral imaging of dynamic samples. Additionally, since all the spectral images are captured simultaneously, the method has potential for studying samples prone to photobleaching. Finally, this application of FRAME makes it possible to discriminate between signals due to infinitely spectrally close sources which, to the best of the authors' knowledge, has not been possible in snapshot multispectral imaging schemes before.