Bares Amanda J, Mejooli Menansili A, Pender Mitchell A, Leddon Scott A, Tilley Steven, Lin Karen, Dong Jingyuan, Kim Minsoo, Fowell Deborah J, Nishimura Nozomi, Schaffer Chris B
The Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
Center for Vaccine Biology and Immunology, Dept. of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Optica. 2020 Nov 20;7(11):1587-1601. doi: 10.1364/optica.389982.
The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally-distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, . We demonstrated the utility of this instrument for spectral separation of closely-overlapped fluorophores in samples containing ten different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple preparations in mouse cortex and inflamed skin with up to eight different cell types or tissue structures distinguished.
多光子显微镜对光学散射不敏感，能够对组织深处进行高分辨率、高对比度成像，包括在活体动物中。然而，散射确实严重限制了光谱色散技术用于提高光谱分辨率。实际上，这种有限的光谱分辨率以及需要多个激发波长来激发不同荧光团，使得多光子显微镜一次只能对少数几个光谱上不同的荧光标记进行成像，限制了可研究的生物过程的复杂性。在这里，我们展示了一种高光谱多光子显微镜，它利用三种不同波长的激发源以及使用角度调谐带通滤波器的多路复用荧光发射检测。这种显微镜保持对散射不敏感，同时在发射的荧光上提供足够高的光谱分辨率，并利用荧光染料的波长依赖性非线性激发来实现多个光谱重叠标记的清晰分离。我们展示了该仪器在含有十种不同颜色荧光珠的样品、表达多达七种不同荧光蛋白融合构建体的活细胞以及小鼠皮层和炎症皮肤的多种制剂中对紧密重叠荧光团进行光谱分离的效用，其中可区分多达八种不同的细胞类型或组织结构。
