Multispectral Fluorescence Imaging

Abstract

Multispectral fluorescence imaging (MSFI) is a rapidly growing field with broad applications in both preclinical and clinical settings. Application of this novel technology in small-animal imaging and microscopy produces enhanced sensitivity and reliable quantification and resolves multiple simultaneous signals. MSFI flow cytometry can quantify multiple fluorescent parameters with morphologic or subcellular spatial details on millions of cells. MSFI has the potential to improve the accuracy of disease detection or differentiation and intrasurgical metastatic diagnosis, guide neurosurgeries, and monitor treatment response.Key Words: multispectral fluorescence imaging; multiplexing; spectral unmixing; small-animal imaging; microscopy; flow cytometry Multispectral imaging (MSI) is the synergistic combination of imaging and spectroscopy. Color is the appearance of a light most affected by wavelength (l) and the observers' visual system. Objects with similar colors are not necessarily the same. The simplest example is the spectrally pure yellow and the mixture of red and green, which have completely different spectral content but precisely the same redgreen-blue (RGB) coordinates (1). Spectroscopy is the technique of breaking light down into its composite colors to identify the composition of the object. Distinguished from conventional RGB full-color imaging, MSI can obtain a high-resolution optical spectrum for each image pixel, resulting in a series of images of the same field of view that are acquired at different wavelengths. These can be stacked in 3-dimensional datasets or a cube (x, y, and l). After proper calibration, quantitative images of individual analytes can be generated. Excellent reviews of MSI instrumentation and data analysis exist (1,2). Briefly, a camera is used to acquire the spatial information, and the spectral information is gained by scanning a dispersive element to record spectra for each image. Electronic tunable filters such as an acousto-optic tunable filter and liquid crystal tunable filters (LCTFs) are preferable to mechanically scanning dispersive devices (filter wheels, monochromators) because they are quiet, fast, compact, and stable and demonstrate increased spectral selectivity, spectral purity, and flexibility. The disadvantages of electronic tunable filters include low light throughput, photobleaching, and inappropriate capture if significant sample or camera movement occurs during the acquisition or if high temporal resolution is needed to capture certain events (e.g., calcium signaling transients) (3). The typical method for data analysis of MSI is a least-squares-fitting linear unmixing approach with additional constraints (such as nonnegativity), resulting component images, and a final composite image. This approach is limited to spectral analysis only, whereas combining the rich spatial information in the images with the spectral data is a more powerful and evolving field (1). Multispectral fluorescence imaging (MSFI) coupled with microscopy and flow cytometry are commercial...

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