Massive wavelength-division multiplexing and imaging with laser particles

摘要 荧光是目前活体显微镜和细胞计数的标准选择方法。 然而，荧光探针的宽发射光谱是荧光染料、荧光探针和荧光探针的缺点。 量子点限制了可以同时跟踪而不模糊的细胞的数量。 DNA条形码可以标记细胞，但不能在体内可视化，因为它们需要体外遗传标记。 阅读。激光器中使用的受激发射和腔共振的光学原理可以 在宽光谱范围内产生极窄的光谱线宽。该项目将 将激光放大到线粒体或病毒的大小，并开发仪器来利用 激光粒子作为大规模并行成像和分析的新型探针。通过追踪 在小鼠中，随着时间的推移，单个细胞的增殖、迁移以及细胞-细胞和细胞-组织 可以在体内研究相互作用。可以通过流式细胞术进一步分析细胞， 进行基因分析和单细胞RNA测序，提供全面的信息 从分子、细胞、组织和系统水平，单个细胞中有数百万到数十亿个细胞， 动物实验第一个具体目标是创建一个新的范例， 使用可注射的、生物相容的微腔和亚微米腔激光器进行细胞标记。第二 目的是发展激光粒子受激发射（LASE）显微镜，用于进行标记 显微镜在体内的深度达3毫米。第三个具体目标是证明大规模 多路复用、高通量细胞跟踪和分析。突破能力将是 用于分析肿瘤在进展、转移和治疗反应方面的异质性， 前所未有的单细胞分辨率。

ABSTRACT Fluorescence is the current standard method of choice for intravital microscopy and cytometry. However, the broad emission spectrum of fluorescent probes⎯dyes, fluorescent probes, and quantum dots⎯limits the number of cells that can be tracked simultaneously without ambiguity. DNA barcodes can label cells but cannot be visualized in vivo, as they require in vitro genetic reading. The optical principle of stimulated emission and cavity resonance used in a laser can generate extremely narrow spectral line-widths over a broad spectral range. This project will miniaturize lasers to the sizes of mitochondria or viruses and develop instrumentations to utilize the laser particles as novel probes for massively parallel imaging and assays. By tracing individual cells over time in mice, the proliferation, migrations and cell-cell and cell-tissue interactions can be studied in vivo. The cells can be further analyzed by flow cytometry and sorted for gene profiling and single-cell RNA sequencing, providing comprehensive information from molecular, cellular, tissue, and systems levels over millions to billions of cells in a single animal experiment. The first specific aim is to create a new paradigm for imaging-compatible cellular labeling using injectable, biocompatible micro- and submicron-cavity lasers. The second aim is to develop Laser Particle Stimulated Emission (LASE) Microscopy for conducting labeled microscopy in vivo at depths of up to 3 mm. The third specific aim is to demonstrate massively multiplexed, high-throughput cell tracking and analysis. The breakthrough capabilities will be used to dissect tumor heterogeneity in progression, metastasis, and response to therapy at unprecedented single-cell resolution.