Tribute to Xiaoliang Sunney Xie

Tribute to Xiaoliang Sunney Xi

Observation of Frequency-Domain Fluorescence Anomalous Phase Advance Due to Dark-State Hysteresis

Frequency-domain fluorescence spectroscopy, commonly referred to as phase fluorometry, is a classic approach to study the lifetime dynamics of fluorescent systems. Here we report an interesting phenomenon: unlike conventional fluorescence lifetime phase fluorometry in which the fluorescence trace always lags behind the modulated excitation source, the detected signal from certain fluorophores can actually exhibit fluorescence anomalous phase advance (FAPA) as if the fluorescence is emitted “ahead” of the source. FAPA is pronounced only within a range of modulation frequencies that are outside quasi-static and quasi-equilibrium conditions. We attribute FAPA to photoinduced dark state hysteresis, supported by both simulations of photodynamic transitions and experiments with dark-state promoters and quenchers. Being a fast and straightforward frequency-domain reporter, FAPA offers a unique and specific contrast mechanism for dark state dynamics sensing and imaging

Quantitative Label-Free Chemical Imaging of PLGA Nanoparticles in Cells and Tissues with Single-Particle Sensitivity

Nanomedicine has brought significant advancements to healthcare by utilizing nanotechnology in medicine. Despite much promise, the further development of nanocarriers for clinical use has been hindered by a lack of understanding and visualization of nano-bio interactions. Conventional imaging methods have limitations in resolution, sensitivity, and specificity. This study introduces a label-free optical approach using stimulated Raman scattering (SRS) microscopy to image poly(lactic-co-glycolic acid) (PLGA) nanocarriers, the most widely used polymeric nanocarrier for delivery therapeutic agents, with single-particle sensitivity and quantification capabilities. A unique Raman peak was identified for PLGA ester, enabling generalized bio-orthogonal bond imaging. We demonstrated quantitative SRS imaging of PLGA nanocarriers across different biological systems from cells to animal tissues. This label-free imaging method provides a powerful tool for studying this prevalent nanocarrier and quantitatively visualizing their distribution, interaction, and clearance in vivo

Bioluminescence Assisted Switching and Fluorescence Imaging (BASFI)

Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are two major biophysical techniques for studying nanometer-scale motion dynamics within living cells. Both techniques read photoemission from the transient RET-excited acceptor, which makes RET and detection processes inseparable. We here report a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI) using a bioluminescent <i>Renilla</i> luciferase RLuc8 as the donor and a photochromic fluorescent protein Dronpa as the acceptor. When in close proximity, RET from RLuc8 switches Dronpa from its original dark state to a stable bright state, whose fluorescence is imaged subsequently with an external laser. Such decoupling between RET and imaging processes in BASFI promises high photon flux as in FRET and minimal bleedthroughs as in BRET. We demonstrated BASFI with Dronpa-RLuc8 fusion constructs and drug-inducible intermolecular FKBP-FRB protein–protein interactions in live cells with high sensitivity, resolution, and specificity. Integrating the advantages of FRET and BRET, BASFI will be a valuable tool for various biophysical studies

Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

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. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH₃) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice <i>in vivo</i>. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans

Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette

Vibrational imaging such as Raman microscopy is a powerful technique for visualizing a variety of molecules in live cells and tissues with chemical contrast. Going beyond the conventional label-free modality, recent advance of coupling alkyne vibrational tags with stimulated Raman scattering microscopy paves the way for imaging a wide spectrum of alkyne-labeled small biomolecules with superb sensitivity, specificity, resolution, biocompatibility, and minimal perturbation. Unfortunately, the currently available alkyne tag only processes a single vibrational “color”, which prohibits multiplex chemical imaging of small molecules in a way that is being routinely practiced in fluorescence microscopy. Herein we develop a three-color vibrational palette of alkyne tags using a ¹³C-based isotopic editing strategy. We first synthesized ¹³C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-¹³C (¹³C¹²C) and bis-¹³C (¹³C¹³C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled (¹²C¹²C) alkynyl probe. We further demonstrated three-color chemical imaging of nascent DNA, RNA, and newly uptaken fatty-acid in live mammalian cells with a simultaneous treatment of three different isotopically edited alkynyl metabolic reporters. The alkyne vibrational palette presented here thus opens up multicolor imaging of small biomolecules, enlightening a new dimension of chemical imaging

Determination of the Subcellular Localization and Mechanism of Action of Ferrostatins in Suppressing Ferroptosis

Ferroptosis is a form of nonapoptotic cell death characterized by the unchecked accumulation of lipid peroxides. Ferrostatin-1 and its analogs (ferrostatins) specifically prevent ferroptosis in multiple contexts, but many aspects of their molecular mechanism of action remain poorly described. Here, we employed stimulated Raman scattering (SRS) microscopy coupled with small vibrational tags to image the distribution of ferrostatins in cells and found that they accumulate in lysosomes, mitochondria, and the endoplasmic reticulum. We then evaluated the functional relevance of lysosomes and mitochondria to ferroptosis suppression by ferrostatins and found that neither is required for effective ferroptosis suppression