Research Projects

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules.

Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels.

Tumor metabolism supports the abnormal survival and growth of malignant cells by providing energy, biomolecular precursors, and reducing equivalents. It is regulated by a combination of cell-intrinsic and cell-extrinsic factors. Upregulated syntheses of new biomolecules in cancer cells are the hallmark biological characteristic in tumor. However, current techniques such as MRI and PET have limited spatial resolution to observe caner metabolism and its heterogeneity at subcellular resolution.

Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. To visualize glucose uptake activity in live cells and tissues, we are performing SRS imaging on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished.

High detection sensitivity and fine chemical selectivity are of pivotal importance for microscopy. While fluorescence microscopy offers superb sensitivity, it is limited by its relatively poor selectivity. We recently reported electronic pre-resonance stimulated Raman scattering (SRS) microscopy by combining the superb sensitivity of electronic spectroscopy and chemical specificity of vibrational spectroscopy.

Chemical bonds are inherent targets for optical spectroscopy, offering power spectroscopic contrast for chemical imaging. Stimulated Raman scattering (SRS) microscopy has emerged as a highly sensitive and specific vibrational imaging technique. When the energy gap between two lasers (pump beam and Stokes beam) is resonant with the vibrational level of targeted chemical bonds, the joint action of the pump and Stokes fields stimulates (i.e., accelerates) the otherwise slow vibrational transition by 108 times.

With the good specificity, sensitivity, photo-stability and live-cell compatibility, we combined five organelle-targeted Carbow-based Raman probes and five fluorescent reporters to achieve tandem 10-color optical imaging of subcellular structures in live cells (including plasma membrane, ER, Golgi, mitochondria, lysosome, lipid droplets, nucleus, tubulin and actin). Here the unprecedented 10-color organelle imaging in live cells doesn’t require any unmixing or color compensation in image processing, which is extremely difficult to achieve by other means.

Lipids are a broad class of vital biomolecules. Together they play indispensable roles in energy storage, membrane architecture and cell signaling. However, elevated lipid concentration can pose detrimental effects leading to cell death. Such aberrant lipid biology is quite general, and contributes to metabolic disorders such as obesity and diabetes, as it has been well documented in a wide spectrum of cells and tissues. Importantly, saturated fatty acids (SFAs), but not unsaturated fatty acids (UFAs), can elicit the lipotoxicity. Mysteriously, UFAs even reverse the impairments of SFAs.

Systems biology and personalized medicine demand high-throughput analysis of cells and biomolecules such as antigens and drugs, the core technology of which often requires distinguishable barcoding. We hence applied Carbow to optical data storage and identification on microbeads. 10 resolvable frequencies at 3 distinct intensity levels (i.e., ternary digit) readily renders 310-1=59048 distinct barcodes, yielding the largest number of distinct spectral barcodes to date, whereas the literature record is around 1000.

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems.

We synthesized a novel super-multiplexed palette of Raman probes, by engineering conjugated poly-ynes (also called carbon-atom wires). Compared to other carbon materials such as graphene and carbon nanotube, carbon-atom wire is one of the least studied carbon allotropes with 1-D structure. We realized and harnessed the optical tunability of these wires. By meticulously engineering of the conjugation length, isotope doping and end-capping substitutions, we synthesized a library of wires with distinct Raman frequencies. 20 of them are termed as Carbon rainbow (i.e., Carbow).

Modern science and technology have exploding demands for multiplexing techniques to simultaneously measure a large number of distinctive species. Notable scientific research areas include exploring structure–function relationships in brain, in situ RNA profiling for spatial transcriptome; understanding heterogeneity of tumor microenvironments; revealing intricate organelle interactions of living cells.

It is extremely desirable to be able to probe biological activities deep inside living organisms. Utilizing a nonlinear excitation scheme, two-photon fluorescence is the most successful optical microscopy for this endeavor. However, a fundamental imaging depth limit still exists for two-photon fluorescence microscopy when imaging highly scattering samples, accompanied by the inevitable background excitation. Essentially, the optical sectioning picture breaks down when approaching the fundamental depth limit.

Z. Zhao, Y. Shen, F. Hu and W. Min. “Applications of vibrational tags in biological imaging by Raman microscopy”, Analyst, 142, 4018 (2017).

F. Hu, S. D. Brucks, T. Lambert, L. Campos and W. Min. Stimulated Raman scattering of polymer nanoparticles for multiplexed live-cell imaging. Chem. Commun., 53, 6187 (2017).

Z. Chen, D. Paley, L. Wei, A. Weisman, R. Friesner, C. Nuckolls* and W. Min*. “Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette”, J. Am. Chem. Soc. 136, 8027 (2014).