Wei Min - Cochemist

Stimulated Raman scattering of polymer nanoparticles for multiplexed live-cell imaging

Co-reporter:Fanghao Hu;Spencer D. Brucks;Tristan H. Lambert;Luis M. Campos

Chemical Communications 2017 vol. 53(Issue 46) pp:6187-6190

Publication Date(Web):2017/06/06

DOI:10.1039/C7CC01860F

A novel nanoparticle-based imaging strategy is introduced that couples biocompatible organic nanoparticles and stimulated Raman scattering (SRS) microscopy. Polymer nanoparticles with vibrational labels incorporated were readily prepared for multi-color SRS imaging with excellent photo-stability. The Raman-active polymer dots are nontoxic, rapidly enter various cell types, and are applied in multiplexed cell-type sorting.

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Co-reporter:Yihui Shen;Zhilun Zhao;Luyuan Zhang;Lingyan Shi;Sanjid Shahriar;Robin B. Chan;Gilbert Di Paolo

PNAS 2017 114 (51 ) pp:13394-13399

Publication Date(Web):2017-12-19

DOI:10.1073/pnas.1712555114

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Co-reporter:Yihui Shen;Zhilun Zhao;Luyuan Zhang;Lingyan Shi;Sanjid Shahriar;Robin B. Chan;Gilbert Di Paolo

PNAS 2017 114 (51 ) pp:13394-13399

Publication Date(Web):2017-12-19

DOI:10.1073/pnas.1712555114

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Co-reporter:Yihui Shen;Zhilun Zhao;Luyuan Zhang;Lingyan Shi;Sanjid Shahriar;Robin B. Chan;Gilbert Di Paolo

PNAS 2017 114 (51 ) pp:13394-13399

Publication Date(Web):2017-12-19

DOI:10.1073/pnas.1712555114

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Metabolic activity induces membrane phase separation in endoplasmic reticulum

Co-reporter:Yihui Shen;Zhilun Zhao;Luyuan Zhang;Lingyan Shi;Sanjid Shahriar;Robin B. Chan;Gilbert Di Paolo

PNAS 2017 114 (51 ) pp:13394-13399

Publication Date(Web):2017-12-19

DOI:10.1073/pnas.1712555114

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Applications of vibrational tags in biological imaging by Raman microscopy

Co-reporter:Zhilun Zhao;Yihui Shen;Fanghao Hu

Analyst (1876-Present) 2017 vol. 142(Issue 21) pp:4018-4029

Publication Date(Web):2017/10/23

DOI:10.1039/C7AN01001J

As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman microscopy has made a big impact in many disciplines of science. While label-free imaging has been the prevailing strategy in Raman microscopy, recent development and applications of vibrational/Raman tags, particularly when coupled with stimulated Raman scattering (SRS) microscopy, have generated intense excitement in biomedical imaging. SRS imaging of vibrational tags has enabled researchers to study a wide range of small biomolecules with high specificity, sensitivity and multiplex capability, at a single live cell level, tissue level or even in vivo. As reviewed in this article, this platform has facilitated imaging distribution and dynamics of small molecules such as glucose, lipids, amino acids, nucleic acids, and drugs that are otherwise difficult to monitor with other means. As both the vibrational tags and Raman instrumental development progress rapidly and synergistically, we anticipate that this technique will shed light onto an even broader spectrum of biomedical problems.

Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes

Co-reporter:Lu Wei, Fanghao Hu, Zhixing Chen, Yihui Shen, Luyuan Zhang, and Wei Min

Accounts of Chemical Research 2016 Volume 49(Issue 8) pp:1494

Publication Date(Web):August 3, 2016

DOI:10.1021/acs.accounts.6b00210

ConspectusInnovations 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. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy.To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of “omics”.As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenografts, and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species.

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

Co-reporter:Lu Wei, Yihui Shen, Fang Xu, Fanghao Hu, Jamie K. Harrington, Kimara L. Targoff, and Wei Min

ACS Chemical Biology 2015 Volume 10(Issue 3) pp:901

Publication Date(Web):January 5, 2015

DOI:10.1021/cb500787b

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 in vivo. 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.

Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Co-reporter:Fanghao Hu;Dr. Zhixing Chen;Dr. Luyuan Zhang;Yihui Shen;Lu Wei; Wei Min

Angewandte Chemie International Edition 2015 Volume 54( Issue 34) pp:9821-9825

Publication Date(Web):

DOI:10.1002/anie.201502543

Abstract

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. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell–cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution.

Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Co-reporter:Fanghao Hu;Dr. Zhixing Chen;Dr. Luyuan Zhang;Yihui Shen;Lu Wei; Wei Min

Angewandte Chemie 2015 Volume 127( Issue 34) pp:9959-9963

Publication Date(Web):

DOI:10.1002/ange.201502543

Abstract

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. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell–cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution.

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

Co-reporter:Zhixing Chen ; Daniel W. Paley ; Lu Wei ; Andrew L. Weisman ; Richard A. Friesner ; Colin Nuckolls

Journal of the American Chemical Society 2014 Volume 136(Issue 22) pp:8027-8033

Publication Date(Web):May 21, 2014

DOI:10.1021/ja502706q

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.

Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling

Co-reporter:Fanghao Hu, Lu Wei, Chaogu Zheng, Yihui Shen and Wei Min

Analyst 2014 vol. 139(Issue 10) pp:2312-2317

Publication Date(Web):20 Jan 2014

DOI:10.1039/C3AN02281A

Choline is a small molecule that occupies a key position in the biochemistry of all living organisms. Recent studies have strongly implicated choline metabolites in cancer, atherosclerosis and nervous system development. To detect choline and its metabolites, existing physical methods such as magnetic resonance spectroscopy and positron emission tomography are often limited by the poor spatial resolution and substantial radiation dose. Fluorescence imaging, although with submicrometer resolution, requires introduction of bulky fluorophores and thus is difficult in labeling the small choline molecule. By combining the emerging bond-selective stimulated Raman scattering microscopy with metabolic incorporation of deuterated choline, herein we have achieved high resolution imaging of choline-containing metabolites in living mammalian cell lines, primary hippocampal neurons and the multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal a functional difference in the choline metabolism and lipid-mediated signaling events. In neurons, choline incorporation is visualized within both soma and neurites, where choline metabolites are more evenly distributed compared to proteins. Furthermore, choline localization is also observed in the pharynx region of C. elegans larvae, consistent with its organogenesis mechanism. These applications demonstrate the potential of isotope-based stimulated Raman scattering microscopy for future choline-related disease detection and development monitoring in vivo.

Live-Cell Quantitative Imaging of Proteome Degradation by Stimulated Raman Scattering

Co-reporter:Yihui Shen;Fang Xu;Lu Wei;Fanghao Hu; Wei Min

Angewandte Chemie International Edition 2014 Volume 53( Issue 22) pp:5596-5599

Publication Date(Web):

DOI:10.1002/anie.201310725

Abstract

Protein degradation is a regulatory process essential to cell viability and its dysfunction is implicated in many diseases, such as aging and neurodegeneration. In this report, stimulated Raman scattering microscopy coupled with metabolic labeling with ¹³C-phenylalanine is used to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous ¹²C-phenylalanine and incorporated ¹³C-phenylalanine as protein markers for the original and nascent proteomes, respectively, and the decay of the former wasquantified through ¹²C/(¹²C+¹³C) ratio maps. We demonstrate time-dependent imaging of proteomic degradation in mammalian cells under steady-state conditions and various perturbations, including oxidative stress, cell differentiation, and huntingtin protein aggregation.

Live-Cell Quantitative Imaging of Proteome Degradation by Stimulated Raman Scattering

Co-reporter:Yihui Shen;Fang Xu;Lu Wei;Fanghao Hu; Wei Min

Angewandte Chemie 2014 Volume 126( Issue 22) pp:5702-5705

Publication Date(Web):

DOI:10.1002/ange.201310725

Abstract

Protein degradation is a regulatory process essential to cell viability and its dysfunction is implicated in many diseases, such as aging and neurodegeneration. In this report, stimulated Raman scattering microscopy coupled with metabolic labeling with ¹³C-phenylalanine is used to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous ¹²C-phenylalanine and incorporated ¹³C-phenylalanine as protein markers for the original and nascent proteomes, respectively, and the decay of the former wasquantified through ¹²C/(¹²C+¹³C) ratio maps. We demonstrate time-dependent imaging of proteomic degradation in mammalian cells under steady-state conditions and various perturbations, including oxidative stress, cell differentiation, and huntingtin protein aggregation.

Bioluminescence Assisted Switching and Fluorescence Imaging (BASFI)

Co-reporter:Luyuan Zhang, Fang Xu, Zhixing Chen, Xinxin Zhu, and Wei Min

The Journal of Physical Chemistry Letters 2013 Volume 4(Issue 22) pp:3897-3902

Publication Date(Web):November 1, 2013

DOI:10.1021/jz402128j

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 Renilla 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.Keywords: bioluminescence; energy transfer; live-cell imaging; photoswitching; protein−protein interaction;

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

Co-reporter:Evangelos Gatzogiannis, Zhixing Chen, Lu Wei, Richard Wombacher, Ya-Ting Kao, Grygorii Yefremov, Virginia W. Cornish and Wei Min

Chemical Communications 2012 vol. 48(Issue 69) pp:8694-8696

Publication Date(Web):06 Jul 2012

DOI:10.1039/C2CC33133K

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.

Molecular-Switch-Mediated Multiphoton Fluorescence Microscopy with High-Order Nonlinearity

Co-reporter:Xinxin Zhu, Ya-Ting Kao, and Wei Min

The Journal of Physical Chemistry Letters 2012 Volume 3(Issue 15) pp:2082-2086

Publication Date(Web):July 25, 2012

DOI:10.1021/jz300607c

Two-photon excited fluorescence microscopy is now an indispensable imaging tool for studying biological samples because of its intrinsic optical sectioning. However, both of its contrast and penetration depth are still limited when imaging deep inside of scattering samples. Herein, we propose a general spectroscopy concept to enhance the image contrast and the fundamental depth limit of two-photon imaging. We show that the population transfer kinetics of the photoinduced molecular switches could generate additional high-order nonlinearity between the signal and the laser intensity. Due to the long-lived nature of these switchable states, the incident photons can operate in a sequential manner, and the nonlinearity effect could accumulate (up to sixth order) as the population is being cycled through these states. Conceptually different from conventional multiphoton processes mediated by transient virtual states, our strategy constitutes a new class of fluorescence microscopy with high-order nonlinearity that is mediated by population transfer.Keywords: nonlinear microscopy; optical highlighters; photoactivatable protein; photoswitchable protein; photoswitches; super-resolution; two-photon fluorescence microscopy;

Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein

Co-reporter:Ya-Ting Kao;Xinxin Zhu

PNAS 2012 Volume 109 (Issue 9 ) pp:

Publication Date(Web):2012-02-28

DOI:10.1073/pnas.1115311109

Recent advances in fluorescent proteins (FPs) have generated a remarkable family of optical highlighters with special light responses. Among them, Dronpa exhibits a unique capability of reversible light-regulated on-off switching. However, the environmental dependence of this photochromism is largely unexplored. Herein we report that the photoswitching kinetics of the chromophore inside Dronpa is actually slowed down by increasing medium viscosity outside Dronpa. This finding is a special example of an FP where the environment can exert a hydrodynamic effect on the internal chromophore. We attribute this effect to protein-flexibility mediated coupling where the chromophore’s cis-trans isomerization during photoswitching is accompanied by conformational motion of a part of the protein β-barrel whose dynamics should be hindered by medium friction. Consistent with this mechanism, the photoswitching kinetics of Dronpa-3, a structurally more flexible mutant, is found to exhibit a more pronounced viscosity dependence. Furthermore, we mapped out spatial distributions of microviscosity in live cells experienced by a histone protein using the photoswitching kinetics of Dronpa-3 fusion as a contrast mechanism. This unique reporter should provide protein-specific information about the crowded intracellular environments by offering a genetically encoded microviscosity probe, which did not exist with normal FPs before.

Pump-probe optical microscopy for imaging nonfluorescent chromophores

Co-reporter:Lu Wei

Analytical and Bioanalytical Chemistry 2012 Volume 403( Issue 8) pp:2197-2202

Publication Date(Web):2012 June

DOI:10.1007/s00216-012-5890-1

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light–molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

Frequency-domain phase fluorometry in the presence of dark states: A numerical study

Co-reporter:Xinxin Zhu, Wei Min

Chemical Physics Letters 2011 Volume 516(1–3) pp:40-44

Publication Date(Web):7 November 2011

DOI:10.1016/j.cplett.2011.09.054

Abstract

Fluorescence anomalous phase advance (FAPA) is a newly discovered spectroscopy phenomenon: instead of lagging behind the modulated light, fluorescence signal can exhibit FAPA as if it precedes the excitation source in time. While FAPA offers a promising technique for probing dark state lifetime, the underlying mechanism is not fully elucidated. Herein we investigate frequency-domain phase fluorometry as a result of intricate interplay between a short-lived fluorescent state and a long-lived dark state. In particular, the quantitative dependence on modulation frequency, excitation intensity, nonradiative decay, intersystem crossing and dark-state lifetime are explored respectively. A comprehensive view of phase fluorometry emerges consequently.

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

Co-reporter:Evangelos Gatzogiannis, Xinxin Zhu, Ya-Ting Kao, and Wei Min

The Journal of Physical Chemistry Letters 2011 Volume 2(Issue 5) pp:461-466

Publication Date(Web):February 14, 2011

DOI:10.1021/jz2000134

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.Keywords: dark state; fluorescence lifetime; frequency-domain fluorescence lifetime measurement; phase fluorometry; triplet state;

Stimulated Raman scattering of polymer nanoparticles for multiplexed live-cell imaging

Co-reporter:Fanghao Hu, Spencer D. Brucks, Tristan H. Lambert, Luis M. Campos and Wei Min

Chemical Communications 2017 - vol. 53(Issue 46) pp:NaN6190-6190

Publication Date(Web):2017/04/25

DOI:10.1039/C7CC01860F

A novel nanoparticle-based imaging strategy is introduced that couples biocompatible organic nanoparticles and stimulated Raman scattering (SRS) microscopy. Polymer nanoparticles with vibrational labels incorporated were readily prepared for multi-color SRS imaging with excellent photo-stability. The Raman-active polymer dots are nontoxic, rapidly enter various cell types, and are applied in multiplexed cell-type sorting.

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

Co-reporter:Evangelos Gatzogiannis, Zhixing Chen, Lu Wei, Richard Wombacher, Ya-Ting Kao, Grygorii Yefremov, Virginia W. Cornish and Wei Min

Chemical Communications 2012 - vol. 48(Issue 69) pp:NaN8696-8696

Publication Date(Web):2012/07/06

DOI:10.1039/C2CC33133K

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.