Molecular Holography: A 3D Highly Multiplexed Alternative to Fluorescence Microscopy

动机细胞生物学的突破性进展很快伴随着新的成像方式的发展。几十年来，荧光显微镜一直是生物医学研究的基本工具。传统的显微镜技术捕获二维图像;然而，由于细胞及其相互作用是动态的三维系统，传统的荧光显微镜技术通过线或点扫描序列在二维中映射来捕获这些图像，然后堆叠以创建3D图片。数字内联全息显微镜（DHIM）能够一次性以视频速率对立方毫米体积进行三维成像，无需扫描或堆叠。就像从串行处理器转移到并行处理器一样，全息方法将扫描时间减少了几个数量级。 具有拉曼散射光的全息显微术将创建“分子全息术”，这是一种新技术，其将允许（i）增强的多路复用和（ii）在真实的时间中对许多生物分子（蛋白质、脂质等）进行一次性快速3D跟踪，为生物医学研究创建令人兴奋的新功能成像模式。 目标.这项工作的目标是实现分子全息术，其有望应用于诸如真实的实时3D并行监测多个蛋白质运输生物标志物和其他感兴趣的细胞基质（例如，用于纳米治疗）。 理论与方法。全息图是当来自物体的相干散射光与来自同一光源（即激光器）的光发生干涉时产生的干涉图案。振幅和相位都被记录，可以通过计算重建来恢复体积中任何位置的图像，从而允许三维系统的动态快照。休伊特实验室已经清楚地检测到连续波受激表面增强拉曼散射（cwSESRS）在微瓦级，成功地证明了相干拉曼信号。展望未来，我们将优化这种强烈的cwSESRS信号，并研究已知的脉冲SESRS的线形差异，准备应用于全息技术。 结果。分子全息术的发展将为基础细胞生物学研究创造一种尖端的分析技术，推动知识的重大进步。我们预见这项技术将成为科学家研究复杂和动态3D生物系统（例如细胞内蛋白质转运）的工具包的一个有价值的补充。与荧光显微镜相比，它有几个优点，如极端多路复用，简单的标记和快速的三维采集（并行处理）。我们未来的步骤之一是追求无标记的分子全息术，使用SRS瞄准在大多数蛋白质中发现的~1000 cm-1拉曼苯丙氨酸模式。

Motivation. Breakthroughs in cell biology have quickly followed from the development of new imaging modalities. For several decades, fluorescence microscopy has been a fundamental tool for biomedical research. Traditional microscopy techniques capture two dimensional images; however, because cells and their interactions are dynamic three-dimensional systems, traditional fluorescence microscopy techniques capture these images by mapping in two dimensions through line or point scan sequences, then stacked to create a 3D picture. Digital inline holographic microscopy (DHIM) enables three-dimensional imaging over cubic millimeter volumes at video rates in one shot - with no need for scanning or stacking. Like moving from serial to parallel processors, the holographic approach reduces scan times by several orders of magnitude. Holographic microscopy with Raman scattered light would create "molecular holography", a novel technique which would allow for (i) enhanced multiplexing and (ii) one-shot, fast 3D tracking of many biomolecules (proteins, lipids, etc) in real time, creating an exciting new functional imaging modality for biomedical research. Objectives. The goal of this work is to achieve molecular holography, which promises applications such as real time 3D parallel monitoring of multiple protein trafficking biomarkers, and other cellular substrates of interest (e.g. for nanotherapeutics). Theory and Approach. Holograms are interference patterns produced when the coherent scattered light from an object interferes with light from the same source (i.e. a laser). Both amplitude and phase are recorded, which can be computationally reconstructed to recover images from any location in the volume, allowing dynamic snapshots of the system in three dimensions. The Hewitt lab has clearly detected continuous wave stimulated surface enhanced Raman scattering (cwSESRS) at microwatt levels, successfully demonstrating a coherent Raman signal. Moving forward, we will optimize this intense cwSESRS signal and investigate known differences in lineshape from pulsed SESRS, preparing the technique for application to holography. Outcome. Development of molecular holography will create a cutting-edge analytical technique for fundamental cell biology research, driving significant advancement of knowledge. We foresee this technique becoming a valuable addition to scientists' toolkit for studying complex and dynamic 3D biological systems, for example intracellular protein transport. It has several advantages over fluorescence microscopy, such as extreme multiplexing, simple labeling, and rapid three-dimensional acquisition (parallel processing). One of our future steps is the pursuit of label-free molecular holography, using SRS to target the ~1000 cm-1 Raman phenylalanine mode found in most proteins.