Chromatic two-photon fluorescence microsopy using band-shifting imaging probes

使用频移成像探针的彩色双光子荧光显微镜 双光子荧光显微镜已经成为最重要的成像手段之一 生物研究和医学诊断。尽管存在许多实现高速双光子的技术 在横向成像时，轴向成像速度往往受到机械扫描速度慢的限制 物镜或标本，这对快速生物监测提出了重大挑战 多深度处理以及3D处理和微型内窥镜的发展。要克服这一点 本文提出了一种基于频移的光谱编码双光子成像新技术 成像探头，可实现平行轴向成像。具体来说，不同的激发波长是 聚焦到不同的轴向位置(通过特意引入的色差)来激发双光子 荧光来自频移成像探头，它在激发时移动发射频带 波长各不相同。因此，对不同轴向位置处的荧光信号进行频谱编码以 显示不同的光谱带，因此可以使用分光计或阵列并行成像 波长分辨率探测器。建议的频移成像探针将被合成、优化、 并用于细胞标记。对分子结构、分子质量、 将进行光物理和双光子性质的研究。提出的彩色双光子成像 系统将进行设计、开发和优化。包括映射关系的系统指标 在轴向位置和荧光波段之间移动时，轴向成像范围和空间分辨率会有所不同 特色化的。所提出的方法将通过对细胞和 组织模型以及结直肠癌异种移植模型的体内成像研究。

Chromatic two-photon fluorescence microscopy using band-shifting imaging probes Two-photon fluorescence microscopy has emerged as one of the most important imaging modalities for biological research and medical diagnosis. Although many techniques exist for realizing high speed two-photon imaging in the lateral directions, the axial imaging speed is still often limited by the slow mechanical scanning of the objective lens or the specimen, presenting a significant challenge for monitoring fast biological processes at multiple depths as well as in 3D and the development of miniature endoscopy. To overcome this limitation, here a new spectrally encoded two-photon imaging technique is proposed using band-shifting imaging probes, which can enable parallel axial imaging. Specifically, different excitation wavelengths are focused onto different axial positions (through purposely introduced chromatic aberration) to excite two-photon fluorescence from the band-shifting imaging probes, which shift the emission band when the excitation wavelength varies. As such, the fluorescence signals at different axial positions are spectrally encoded to exhibit different spectral bands, and can thus be imaged in parallel by using a spectrometer or arrayed wavelength-resolving detectors. The proposed band-shifting imaging probes will be synthesized, optimized, and used for cellular labeling. Systematic characterization on the molecular structures, molecular weight, photophysical and two-photon properties will be performed. The proposed chromatic two-photon imaging system will be designed, developed, and optimized. System metrics including the mapping relationship between the axial position and fluorescence band shift, the axial imaging range, and spatial resolutions will be characterized. The proposed method will be demonstrated and validated by performing imaging of cells and tissue phantoms as well as by in vivo imaging studies of a colorectal cancer xenograft model.