Super-multiplex optical imaging of neurons and neural circuits

The ability to simultaneously visualize a large number of molecular species in cells and tissues is indispensable for understanding complex systems such as the brain. Consequently, the development of novel optical methods is one of the major challenges undertaken by the White House BRAIN Initiative. However, this is an extremely difficult task because of the fundamental Òcolor barrierÓ of the fluorescence imaging techniques, as only 2~5 fluorescent colors can be resolved at a time. To address this grand challenge, we have recently developed a library of spectrally ultra-sharp Raman probes and invented a novel Raman spectroscopic imaging technique to break such a Òcolor barrierÓ (Nature 2017; Nature Methods 2018). Using them, we have demonstrated up to 24-color imaging for biological samples including neuron and brain slices. Yet, the instrumentation of the first-generation technology is intrinsically slow and low-throughput, which is largely limited by the narrowband laser source in our laboratory. To break this barrier even further, we propose to acquire a new broadband laser source to build a hyperspectral stimulated Raman scattering (SRS) microscope to perform high-speed super-multiplex optical imaging in neuron and brain tissues. More than 1,000 times faster speed is expected than the first-generation technology. Thus the proposed high-speed super-multiplex imaging technology, together with the continuing development of third-generation Raman probes (up to 50~100 colors), would directly impact many efforts within Columbia UniversityÕs DoD funded MURI that will greatly advance our goals of imaging structural and functional connectivity in neural networks. In particular, this tool will enable exploration of three impactful areas: 1) Molecular Connectome in fixed brain preparations, which would complement the standard electron microscopy based Anatomical Connectome, 2) time-lapse monitoring organelle interactome in live cells; and (3) profiling different cell subtypes in living neurons and slices. By enhancing the numbers of cells and molecules that can be simultaneously imaged, this method could potentially revolutionize optical imaging in biology.