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, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

中文翻译：

---

高光谱多光子显微镜用于多个光谱重叠荧光标记的体内可视化

多光子显微镜对光学散射不敏感，能够对组织深处（包括活体动物）进行高分辨率、高对比度成像。然而，散射确实严重限制了使用光谱色散技术来提高光谱分辨率。在实践中，这种有限的光谱分辨率加上需要多个激发波长来激发不同的荧光团，限制了多光子显微镜一次只能对几个光谱不同的荧光标记进行成像，从而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重荧光发射检测。该显微镜保持了散射不敏感性，同时对发射的荧光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，从而能够在体内清晰分离多个光谱重叠的标记。我们展示了该仪器对包含 10 种不同颜色荧光珠的样品、表达多达 7 种不同荧光蛋白融合结构的活细胞以及小鼠皮质和发炎皮肤的多种体内制剂中紧密重叠的荧光团的光谱分离的实用性，最多可区分八种不同的细胞类型或组织结构。