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

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.