Abstract
Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability.
Highlights
Hyperspectral imaging in microscopy is a powerful method for quantifying the functional and morphological states of cells and tissues
We presented a new near-common-path interferometer design for wide-field imaging Fourier-transform spectroscopy with phase stability similar to industry standard Sagnac imaging spectrometers
In our design, the maximum field of view (FOV) allowed by the microscope optics can be utilized for spectral imaging
Summary
Hyperspectral imaging in microscopy is a powerful method for quantifying the functional and morphological states of cells and tissues. Multicolor spectral karyotyping of human chromosomes is one example [2] Another is spectral fluorescence resonance energy transfer, which provides more precise and robust measurements of protein– protein interactions [3]. Imaging based on many nonfluorescent analytes utilizing their complex vibronic Raman spectra may be realized using hyperspectral imaging microscopy [4]. For many of these applications, improving imaging throughput is of high interest. Enabling fast hyperspectral imaging in wide-field modalities may open doors to new high-throughput imaging studies of complex biological system where tens to hundreds of constituents may need to be resolved
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