Summary
Biological systems are inherently complex and interrelated, as they organize and function
through a series of hierarchical networks involving multiple interacting components. Hence,
simultaneously visualizing a large number of distinct molecular species inside living cells has
become indispensable for understanding these biological processes in a holistic manner. As we
enter the era of systems biology, such super-multiplex imaging capability will be transformative
across various fields including revealing structure–function relationships in nervous systems;
understanding tumor heterogeneity; studying macromolecules choreography during cell
regulation, as well as revealing intricate interactions among various organelles of living cells.
The goal of this project is to develop a general super-multiplex optical microscopy platform
for simultaneously imaging a large number (more than 20) of specific molecular targets inside
live cells, an important but otherwise intractable goal by conventional methods such as
fluorescence. To do so, we propose to couple the emerging electronic pre-resonance stimulated
Raman scattering (epr-SRS) microscopy, offering nanomolar detection sensitivity and narrow
chemical specificity, with novel vibrational probes consisting of triple-bond-conjugated light-
absorbing dyes. The first-generation technique has been recently published, demonstrating a
record of 24-color imaging in biological systems (L. Wei … W. Min. Nature, 544, 465, 2017).
Moving towards the next-generation technology, we have laid out systematic plans as to
how to crystallize this concept into a much more powerful platform to achieve high-speed, high-
sensitivity, super-multiplex vibrational imaging of specific proteins and organelles in living cells.
We propose to construct new microscope instrumentations to significantly boost the imaging
speed by orders of magnitude (Specific Aim 1), and engineer novel epr-SRS vibrational probes
with expanded color palette, superior detection sensitivity, organelle targeting specificity and
genetic encodability to specific proteins (Specific Aim 2). Accompanied by these technical
developments, we will then apply it to probe systems-level interactions within multiple organelles
and proteins during dynamical processes of cytokinesis and apoptosis (Specific Aim 3).
If successfully implemented, we will establish a transformative imaging platform that could
allow researchers to interrogate an unprecedented large number of bio-molecules in living cells
with superb sensitivity, targeting specificity, labeling versatility, and biocompatibility. The
resulting super-multiplex optical microscopy would find wide applications in unraveling complex
biological systems such as cell biology, neurobiology, immunology, and tumor biology.