@article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/107395, title ="Super-Resolution Label-free Volumetric Vibrational Imaging", author = "Qian, Chenxi and Miao, Kun", month = "January", year = "2021", url = "https://resolver.caltech.edu/CaltechAUTHORS:20210111-140338961", note = "The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. \n\nThis version posted January 9, 2021. \n\nWe thank Xun Wang and Dr. Lilien Voong for fruitful discussions. We are grateful to Can Li and Prof. Marianne Bronner for sharing the zebrafish embryo slices. Chenxi Qian acknowledges the support of the Natural Sciences and Engineering Research Council of Canada (NSERC Postdoctoral Fellowship). Lu Wei acknowledges the support of National Institutes of Health (NIH Director’s New Innovator Award, DP2 GM140919-01), Amgen (Amgen Early Innovation Award) and the start-up funds from California Institute of Technology. \n\nData availability: The authors declare that all data supporting the findings of the present study are available in the article and its supplementary figures and tables, or from the corresponding author upon request. \n\nCode availability: MATLAB code used for PSF determination and Python code for U-Net training and prediction in this paper is available at https://github.com/Li-En-Good/VISTA. \n\nThe authors have declared no competing interest.", revision_no = "11", abstract = "Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. However, super-resolution fluorescence techniques, including both optics-oriented and sample-expansion based, are limited in quantification and throughput especially in tissues from photobleaching or quenching of the fluorophores, and low-efficiency or non-uniform delivery of the probes. Here, we report a general sample-expansion vibrational imaging strategy, termed VISTA, for scalable label-free high-resolution interrogations of protein-rich biological structures with resolution down to 82 nm. VISTA achieves decent three-dimensional image quality through optimal retention of endogenous proteins, isotropic sample expansion, and deprivation of scattering lipids. Free from probe-labeling associated issues, VISTA offers unbiased and high-throughput tissue investigations. With correlative VISTA and immunofluorescence, we further validated the imaging specificity of VISTA and trained an image-segmentation model for label-free multi-component and volumetric prediction of nucleus, blood vessels, neuronal cells and dendrites in complex mouse brain tissues. VISTA could hence open new avenues for versatile biomedical studies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/105346, title ="Visualizing Subcellular Enrichment of Glycogen in Live Cancer Cells by Stimulated Raman Scattering", author = "Lee, Dongkwan and Du, Jiajun", journal = "Analytical Chemistry", volume = "92", number = "19", pages = "13182-13191", month = "October", year = "2020", doi = "10.1021/acs.analchem.0c02348", issn = "0003-2700", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200911-133137005", note = "© 2020 American Chemical Society. \n\nReceived: June 1, 2020; Accepted: September 9, 2020; Published: September 9, 2020. \n\nWe thank Dr. Antoni Ribas for sharing the melanoma cell lines. We also thank Dr. Otto Baba and Dr. Morita for sharing the antiglycogen antibodies. We thank Dr. C. Qian, K. Miao, X. Bi, L. Lin, and Dr. L. Voong for helpful discussions. We acknowledge the following agencies and foundations for support: NIH Grant U01 CA217655 (to J.R.H.), the WA State Andy Hill CARE Foundation (to J.R.H.), and an ISB Innovator Award (Y.S.). L.W. acknowledges the support for start-up funds from California Institute of Technology. \n\nThe authors declare no competing financial interest.", revision_no = "17", abstract = "Glycogen, a branched glucose polymer, helps regulate glucose homeostasis through immediate storage and release of glucose. Reprogramming of glycogen metabolism has recently been suggested to play an emerging role in cancer progression and tumorigenesis. However, regulation of metabolic rewiring for glycogen synthesis and breakdown in cancer cells remains less understood. Despite the availability of various glycogen detection methods, selective visualization of glycogen in living cells with high spatial resolution has proven to be highly challenging. Here, we present an optical imaging strategy to visualize glycogen in live cancer cells with minimal perturbation by combining stimulated Raman scattering microscopy with metabolic incorporation of deuterium-labeled glucose. We revealed the subcellular enrichment of glycogen in live cancer cells and achieved specific glycogen mapping through distinct spectral identification. Using this method, different glycogen metabolic phenotypes were characterized in a series of patient-derived BRAF mutant melanoma cell lines. Our results indicate that cell lines manifesting high glycogen storage level showed increased tolerance to glucose deficiency among the studied melanoma phenotypes. This method opens up the possibility for noninvasive study of complex glycogen metabolism at subcellular resolution and may help reveal new features of glycogen regulation in cancer systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/105516, title ="Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma cells", author = "Du, Jiajun and Su, Yapeng", journal = "Nature Communications", volume = "11", pages = "Art. No. 4830", month = "September", year = "2020", doi = "10.1038/s41467-020-18376-x", issn = "2041-1723", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200924-122708002", note = "© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nReceived 21 January 2020; Accepted 14 August 2020; Published 24 September 2020. \n\nL.W. acknowledges start-up fund support from California Institute of Technology. We acknowledge the following agencies and foundations for support: NIH Grant U01 CA217655 (to J.R.H.); the Parker Institute for Cancer Immunotherapy (J.R.H. and A.R.), the WA State Andy Hill CARE Foundation (J.R.H.), and an ISB Innovator Award (Y.S.). \n\nData availability: All the data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request. The following databases are used: Human Reference Genome (UCSC hg 19), Gene Expression Omnibus database (GEO), Molecular Signatures Database (MSigDB). RNA-seq data have been deposited to array express with accession number of E-MTAB-8842. Source data are provided with this paper. \n\nCode availability: Custom code for the surprisal analysis of Raman spectra has previously been published and deposited on GitHub (https://github.com/mesako/Melanoma-Publication) 36. Source data are provided with this paper. \n\nThese authors contributed equally: Jiajun Du, Yapeng Su. \n\nAuthor Contributions: L.W., J.R.H., J.D., and Y.S. conceived the study and designed the experiments. J.D., Y.S., C.Q., D.Y., K.M., D.L., A.N., and R.W. performed the experiments. J.D. and Y.S. analyzed and interpreted the data. L.W., J.R.H., A.R., and R.L. provided conceptual advice on data analysis and interpretation. L.W., J.D., J.R.H., and Y.S. wrote the manuscript. L.W. and J.R.H. supervised this study. \n\nThe authors declare no competing interests. \n\nPeer review information: Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.", revision_no = "20", abstract = "Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/107235, title ="Raman-guided subcellular pharmaco-metabolomics for metastatic melanoma", author = "Du, Jiajun and Su, Yapeng", pages = "PHYS-0320", month = "August", year = "2020", url = "https://resolver.caltech.edu/CaltechAUTHORS:20201221-101511711", note = "© 2020 American Chemical Society.", revision_no = "9", abstract = "We utilized Raman spectro-microscopy to non-invasively probe metabomics within single live cells, aiming to identify\ndruggable metabolic susceptibilities from a series of patient-derived BRAF mutant melanoma cell lines. Each cell line\nrepresents a phenotype with different characteristic level of de-differentiation and BRAFi (BRAF inhibitor) resistance.\nFirst, with single-cell Raman spectroscopy and stimulated Raman scattering (SRS) microscopy, followed by\ntranscriptomics anal., we identified lipid processes as major metabolic functional difference between different\nphenotypes. We then utilized hyperspectral-SRS imaging on intracellular single organelles to identify a previously\nunknown susceptibility of lipid desatn. within de-differentiated cell lines. Drugging this target leads to cellular apoptosis\naccompanied by phase sepd. intracellular domains. The integration of subcellular Raman spectro-microscopy with\nlipidomics and transcriptomics suggests highly heterogenous metabolic responses and possible lipid regulatory\nmechanisms underlying this pharmacol. treatment. Our method should provide a general approach in spatially-resolved\nsingle cell metabolomics studies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/99535, title ="Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling", author = "Miao, Kun and Wei, Lu", journal = "ACS Central Science", volume = "6", number = "4", pages = "478-486", month = "April", year = "2020", doi = "10.1021/acscentsci.9b01196", issn = "2374-7943", url = "https://resolver.caltech.edu/CaltechAUTHORS:20191029-113111264", note = "© 2020 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: November 19, 2019; Published: March 6, 2020. \n\nWe would like to thank Dr. C. Qian, D. Lee, J. Du, and Dr. L. Voong for helpful discussions. We are grateful for the plasmid (mHtt-97Q-GFP) shared by Prof. R. Kopito and Prof. F.-U. Hartl. We thank Prof. Z. Liu for sharing the stable embryonic stem cell-lines. L.W. acknowledges the support of start-up funds from California Institute of Technology. \n\nThe authors declare no competing financial interest.", revision_no = "37", abstract = "Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including in vitro biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington’s disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon–deuterium bonds (C–D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive in vivo investigations.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101868, title ="Chemical probes for optical bio-imaging (Conference Presentation)", author = "Wei, Lu", number = "11256", pages = "Art. No. 112560E", month = "March", year = "2020", doi = "10.1117/12.2551679", isbn = "9781510632752", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200311-151708752", note = "© 2020 Society of Photo-Optical Instrumentation Engineers (SPIE).", revision_no = "5", abstract = "Innovations in novel probes have significantly push the development of new optical spectroscopy and microscopy methods for revealing new information in biological systems. In this talk, I will discuss our recent development by introducing chemical probes to stimulated Raman scattering (SRS) microscopy that could allow multi-functional imaging at sub-cellular level. Both physical and chemical principles underlying the investigation and design of new probes when coupled to the Raman imaging modalities will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101862, title ="High-sensitivity stimulated Raman imaging with chemical tags (Conference Presentation)", author = "Wei, Lu", number = "11250", pages = "Art. No. 112500R", month = "March", year = "2020", doi = "10.1117/12.2548543", isbn = "9781510632639", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200311-151707376", note = "© 2020 Society of Photo-Optical Instrumentation Engineers (SPIE).", revision_no = "5", abstract = "Innovations in optical spectroscopy and microscopy have revolutionized our understanding in biological systems. In this talk, I will discuss our recent development by coupling stimulated Raman scattering (SRS) microscopy with chemical probes that could allow high-sensitivity bio-analysis with fast speed at the sub-cellular level. Both physical and chemical principles underlying the optical microscopy will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101861, title ="Stimulated Raman imaging with chemical probes for subcellular bioanalysis (Conference Presentation)", author = "Wei, Lu and Miao, Kun", number = "11252", pages = "Art. No. 112521S", month = "March", year = "2020", doi = "10.1117/12.2546942", isbn = "9781510632677", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200311-151707274", note = "© 2020 Society of Photo-Optical Instrumentation Engineers (SPIE).", revision_no = "5", abstract = "Innovations in optical spectroscopy and microscopy have revolutionized our understanding in biological systems at sub-cellular levels. In this talk, I will discuss about our recent development by coupling stimulated Raman scattering (SRS) microscopy with chemical probes that could allow new subcellular bioanalysis in live cells. The introduced tags offer additional SRS contrast channel for quantification of biological contents that were previously difficult. Both physical and chemical principles underlying the optical microscopy will be presented, as well as our efforts in biomedical applications including cancer- and neuronal- metabolism.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/97792, title ="Stimulated Raman imaging with chemical probes for subcellular bioanalysis", author = "Wei, Lu", pages = "ANYL-0316", month = "August", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190812-141405591", note = "© 2019 American Chemical Society.", revision_no = "11", abstract = "Innovations in optical spectroscopy and microscopy have revolutionized our understanding in biol. systems at subcellular levels. In this talk, I will present recent advances of two chem. imaging strategies addressing two fundamental challenges in optical bio-imaging. First, we devised a live-cell Bioorthogonal Chem. Imaging platform suited for probing the metabolic dynamics of small bio-mols., which cannot be effectively labeled by bulky fluorophores. This scheme couples the stimulated Raman scattering (SRS) microscopy, a nonlinear vibrational imaging modality that offers rich chem. information, with small vibrational tags including stable isotopes and triple bonds. We applied this platform to a variety of dynamical biol. systems and revealed previously less-known heterogeneity in metabolic signatures governing the obversed cell phenotypes. Second, we developed a supermultiplex optical imaging technique enabled by preresonant SRS microscopy of a newly designed palette of vibrational dyes. This work allows for simultaneous imaging of a large no. of mol. species in live cells and tissues with high sensitivity and specificity, bridging imaging and omics paradigms. Both phys. and chem. principles underlying the optical microscopy will be presented, as well as our recent efforts in biomedical applications including cancer- and neuronal- metab.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/93811, title ="Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy", author = "Wei, Mian and Shi, Lingyan", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "116", number = "14", pages = "6608-6617", month = "April", year = "2019", doi = "10.1073/pnas.1813044116", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190314-130512034", note = "© 2019 National Academy of Sciences. Published under the PNAS license. \n\nEdited by Lance L. Munn, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, and accepted by Editorial Board Member Rakesh K. Jain February 19, 2019 (received for review July 29, 2018). PNAS published ahead of print March 14, 2019. \n\nWe thank E. Silveira for technical assistance; Y. Yang and X. Qu for discussion; and T. Swayne and the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University for help with 3D image analysis, supported by NIH Grant P30 CA013696. W.M. acknowledges support of R01EB020892 from the NIH and the Camille and Henry Dreyfus Foundation. \n\nM.W. and L.S. contributed equally to this work. \n\nAuthor contributions: M.W., L.S., L.W., and W.M. designed research; M.W. and L.S. performed research; Y.S., Z.Z., A.G., and L.J.K. contributed new reagents/analytic tools; M.W. analyzed data; and M.W., L.S., L.W., and W.M. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. L.L.M. is a guest editor invited by the Editorial Board. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1813044116/-/DCSupplemental.", revision_no = "25", abstract = "Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91008, title ="Electronic Resonant Stimulated Raman Scattering Micro-Spectroscopy", author = "Shi, Lixue and Xiong, Hanqing", journal = "Journal of Physical Chemistry B", volume = "122", number = "39", pages = "9218-9224", month = "October", year = "2018", doi = "10.1021/acs.jpcb.8b07037", issn = "1520-6106", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039172", note = "© 2018 American Chemical Society. \n\nReceived: July 22, 2018. Revised: September 8, 2018. Published: September 12, 2018. \n\nPublished as part of The Journal of Physical Chemistry virtual special issue “Young Scientists”. \n\nW.M. acknowledges support from the US Army Research Office (W911NF-12-1-0594) and R01 (EB020892) and the Camille and Henry Dreyfus Foundation. \n\nThe authors declare no competing financial interest.", revision_no = "13", abstract = "Recently we have reported electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy as a powerful technique for super-multiplex imaging (Wei, L.; Nature 2017, 544, 465−470). However, under rigorous electronic resonance, background signal, which mainly originates from pump–probe process, overwhelms the desired vibrational signature of the chromophores. Here we demonstrate electronic resonant stimulated Raman scattering (er-SRS) microspectroscopy and imaging through suppression of electronic background and subsequent retrieval of vibrational peaks. We observed a change of the vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian as the electronic resonance was approached, in agreement with theoretical prediction. In addition, resonant Raman cross sections have been determined after power-dependence study as well as Raman excitation profile calculation. As large as 10^(–23) cm^2 of resonance Raman cross section is estimated in er-SRS, which is about 100 times higher than previously reported in epr-SRS. These results of er-SRS microspectroscopy pave the way for the single-molecule Raman detection and ultrasensitive biological imaging.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91009, title ="Electronic Preresonance Stimulated Raman Scattering Microscopy", author = "Wei, Lu and Min, Wei", journal = "Journal of Physical Chemistry Letters", volume = "9", number = "15", pages = "4294-4301", month = "August", year = "2018", doi = "10.1021/acs.jpclett.8b00204", issn = "1948-7185", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039337", note = "© 2018 American Chemical Society. ACS Editors' Choice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: January 21, 2018. Accepted: July 12, 2018. Published: July 12, 2018. \n\nWe are grateful for discussions with Lixue Shi, Zhixing Chen, Louis Brus, and Sunney Xie. W.M. acknowledges support from NIH Director’s New Innovator Award (1DP2EB016573) and R01 (EB020892) and the Camille and Henry Dreyfus Foundation. \n\nThe authors declare no competing financial interest.", revision_no = "12", abstract = "Optical microscopy has generated great impact for modern research. While fluorescence microscopy provides the ultimate sensitivity, it generally lacks chemical information. Complementarily, vibrational imaging methods provide rich chemical-bond-specific contrasts. Nonetheless, they usually suffer from unsatisfying sensitivity or compromised biocompatibility. Recently, electronic preresonance stimulated Raman scattering (EPR-SRS) microscopy was reported, achieving simultaneous high detection sensitivity and superb vibrational specificity of chromophores. With newly synthesized Raman-active dyes, this method readily breaks the optical color barrier of fluorescence microscopy and is well-suited for supermultiplex imaging in biological samples. In this Perspective, we first review previous utilizations of electronic resonance in various Raman spectroscopy and microscopy. We then discuss the physical origin and uniqueness of the electronic preresonance region, followed by quantitative analysis of the enhancement factors involved in EPR-SRS microscopy. On this basis, we provide an outlook for future development as well as the broad applications in biophotonics.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91010, title ="Operando and three-dimensional visualization of anion depletion and lithium growth by stimulated Raman scattering microscopy", author = "Cheng, Qian and Wei, Lu", journal = "Nature Communications", volume = "9", pages = "Art. No. 2942", month = "July", year = "2018", doi = "10.1038/s41467-018-05289-z", issn = "2041-1723", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039470", note = "© 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. \n\nReceived: 12 December 2017 Accepted: 24 June 2018. Published online: 30 July 2018. \n\nWe acknowledge seed funding support from Columbia University’s Research Initiatives in Science & Engineering competition, started in 2004 to trigger high-risk, high-reward, and innovative collaborations in the basic sciences, engineering, and medicine. Y.Y. acknowledges support from startup funding by Columbia University. W.M. acknowledges support from the US Army Research Office (W911NF-12-1-0594), NIH Director’s New Innovator Award (1DP2EB016573) and R01 (EB020892), and the Camille and Henry Dreyfus Foundation. Z.L and L.-Q.C. acknowledge the support from the Department of Energy, Office of Energy Efficiency and Renewable Energy (EERE), under the Award (DE-EE0007803). Y.Y and Q.C. also want to thank Prof. Alan West at Columbia University for his kind help. \n\nThese authors contributed equally: Qian Cheng, Lu Wei. \n\nAuthor Contributions: Y.Y., W.M., Q.C., and L.W. conceived the idea and designed the experiments. Q.C. and L.W. performed all the experiments and measurements. Z.L, Z.S. and L-Q. C. performed simulations. N.N., B.Z., W.X., M.C., and Y.M. helped prepare and perform experiments. All authors discussed the results. Q.C., L.W., Y.Y., W.M. and Z.L. wrote the paper with the input from all authors. \n\nData availability: The data that support the findings of this study are available from the corresponding author upon reasonable request. \n\nThe authors declare no competing interests.", revision_no = "23", abstract = "Visualization of ion transport in electrolytes provides fundamental understandings of electrolyte dynamics and electrolyte-electrode interactions. However, this is challenging because existing techniques are hard to capture low ionic concentrations and fast electrolyte dynamics. Here we show that stimulated Raman scattering microscopy offers required resolutions to address a long-lasting question: how does the lithium-ion concentration correlate to uneven lithium deposition? In this study, anions are used to represent lithium ions since their concentrations should not deviate for more than 0.1\u2009mM, even near nanoelectrodes. A three-stage lithium deposition process is uncovered, corresponding to no depletion, partial depletion, and full depletion of lithium ions. Further analysis reveals a feedback mechanism between the lithium dendrite growth and heterogeneity of local ionic concentration, which can be suppressed by artificial solid electrolyte interphase. This study shows that stimulated Raman scattering microscopy is a powerful tool for the materials and energy field.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86924, title ="Supermultiplexed optical imaging and barcoding with engineered polyynes", author = "Hu, Fanghao and Zeng, Chen", journal = "Nature Methods", volume = "15", number = "3", pages = "194-200", month = "March", year = "2018", doi = "10.1038/nmeth.4578", issn = "1548-7091", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-125759681", note = "© 2018 Macmillan Publishers Limited. \n\nReceived: 23 August 2017. Accepted: 05 December 2017. Published: 15 January 2018. \n\nWe are grateful for the discussion with L. Brus, Y. Shen and Z. Chen. W.M. acknowledges support from NIH Director's New Innovator Award (1DP2EB016573), R01 (EB020892), the US Army Research Office (W911NF-12-1-0594), and the Camille and Henry Dreyfus Foundation. \n\nAuthor Contributions: F.H. performed the spectroscopy, microscopy and biological studies and analyzed the data with the help of Y.M., L.W. and Q.X.; C.Z. performed the chemical synthesis together with R.L.; F.H. and W.M. conceived the concept; F.H., C.Z. and W.M. designed the experiments and wrote the manuscript with input from all authors. \n\nCompeting interests: Columbia University has filed a patent application (US 62/540,953) based on this study. \n\nCode availability. The MATLAB code is available from the corresponding author upon request. \n\nLife Sciences Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Summary. \n\nData availability. The data that support the findings of this study are provided in Supplementary Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, Supplementary Tables 1–3 and Supplementary Note 1 and are available from the corresponding author upon request.", revision_no = "43", abstract = "Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86916, title ="Super-multiplex vibrational imaging", author = "Wei, Lu and Chen, Zhixing", journal = "Nature", volume = "544", number = "7651", pages = "465-470", month = "April", year = "2017", doi = "10.1038/nature22051", issn = "0028-0836", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-105013735", note = "© 2017 Macmillan Publishers Limited. \n\nreceived 21 September 2016; accepted 3 March 2017. Published online 19 April 2017. \n\nWe thank L. Brus and A. McDermott for discussions, M. Jimenez and C. Dupre for suggestions, and L. Shi for technical assistance. W.M. acknowledges support from an NIH Director’s New Innovator Award (1DP2EB016573), R01 (EB020892), the US Army Research Office (W911NF-12-1-0594), the Alfred P. Sloan Foundation and the Camille and Henry Dreyfus Foundation. R.Y. is supported by the NEI (EY024503, EY011787) and NIMH (MH101218, MH100561). \n\nAuthor Contributions: L.W. carried out the spectroscopy, microscopy and biological studies together with L.S. and with the help of L.Z., F.H. and R.Y.; Z.C. designed and performed chemical synthesis together with R.L., A.V.A. and L.W. under the guidance of V.W.C. and W.M.; L.W. and W.M. conceived the concept; and L.W., Z.C. and W.M. wrote the manuscript with input from all authors. \n\nData availability: All data that support this study are available from the corresponding author on request. Source Data for Fig. 4e are available in the online version of the paper. \n\nCompeting interests: Columbia University has filed a patent application based on this work.", revision_no = "43", abstract = "The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure–function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86919, title ="Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering", author = "Hu, Fanghao and Lamprecht, Michael R.", journal = "Scientific Reports", volume = "6", pages = "Art. No. 39660 ", month = "December", year = "2016", doi = "10.1038/srep39660", issn = "2045-2322", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-112524422", note = "© 2016 The Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ \n\nreceived: 30 September 2016, accepted: 25 November 2016. Published: 21 December 2016. \n\nWe thank Y. Shen and L. Shi for helpful discussions. W. M. acknowledges support from NIH Director’s New Innovator Award (Grant 1DP2EB016573), NIH R01 (Grant EB020892), the US Army Research Office (Grant W911NF-12-1-0594), the Alfred P. Sloan Foundation, and the Camille and Henry Dreyfus Foundation. B.M. acknowledges support from the Army Research Laboratory (Grant W911NF-10-1-0526). M.R.L acknowledges support from a National Science Foundation Graduate Research Fellowship. \n\nAuthor Contributions: F.H., B.M. and W.M. conceived the concept and designed the experiments. F.H., M.R.L. and L.W. performed the experiments. F.H. analyzed the data and wrote the manusript. B.M. and W.M. oversaw the study and edited the manuscript. All authors reviewed the manusript. \n\nThe authors declare no competing financial interests.", revision_no = "19", abstract = "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. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86920, title ="Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes\n", author = "Wei, Lu and Hu, Fanghao", journal = "Accounts of Chemical Research", volume = "49", number = "8", pages = "1494-1502", month = "August", year = "2016", doi = "10.1021/acs.accounts.6b00210", issn = "0001-4842", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-113519551", note = "© 2016 American Chemical Society. \n\nReceived: May 2, 2016. Published: August 3, 2016. \n\nWe appreciate helpful discussions with Meng Wang, Colin Nuckolls, Louis Brus, Ann McDermott, Ronald Breslow, Virginia Cornish, Rafael Yuste, Sunney Xie, and Steven Boxer. This work is supported by NIH Director’s New Innovator Award (Grant 1DP2EB016573), R01 (Grant EB020892), the US Army Research Office (Grant W911NF-12-1-0594), the Alfred P. Sloan Foundation, and the Camille and Henry Dreyfus Foundation. Y. Shen acknowledges support from HHMI International Student Research Fellowship. \n\nThe authors declare the following competing financial interest(s): Columbia University has filed a patent application based on this work.", revision_no = "15", abstract = "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. 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. \n\nTo 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”. \n\nAs 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.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86921, title ="Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy", author = "Hu, Fanhao and Chen, Zhixing", number = "9723", pages = "Art. No. 97230B", month = "April", year = "2016", doi = "10.1117/12.2211787.4848770176001", isbn = "9781628419573", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-114633492", note = "© 2016 Society of Photo-Optical Instrumentation Engineers (SPIE). ", revision_no = "22", abstract = "Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86922, title ="Optical Imaging of Vibrationally-Tagged Small molecules for Biomedicine", author = "Wei, Lu and Min, Wei", pages = "Art. No. BTh2D.2", month = "April", year = "2016", doi = "10.1364/BRAIN.2016.BTh2D.2", isbn = "978-1-943580-10-1", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-115332345", note = "© 2016 Optical Society of America.", revision_no = "10", abstract = "We report a novel imaging platform, by coupling stimulated Raman scattering microscopy with small vibrational tags (including isotopes and alkynes), to probe dynamics of small biomolecules in living organisms with superb sensitivity, specificity and biocompatibility.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86923, title ="Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering", author = "Hu, Fanghao and Chen, Zhixing", journal = "Angewandte Chemie International Edition", volume = "54", number = "34", pages = "9821-9825", month = "August", year = "2015", doi = "10.1002/anie.201502543", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-124956778", note = "© 2015 WILEY‐VCH. \n\nReceived: March 18, 2015. Revised: April 26, 2015. Published online: July 16, 2015. \n\nWe thank Y. Shin for providing hippocampal neurons and J. Hirtz for assistance on mouse brain tissues. W.M. acknowledges support from the NIH Director’s New Innovator Award, ARO MURI W911NF-12-1-0594, and a Alfred P. Sloan Research Fellowship.", revision_no = "15", 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. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86926, title ="Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling", author = "Wei, Lu and Shen, Yihui", journal = "ACS Chemical Biology", volume = "10", number = "3", pages = "901-908", month = "March", year = "2015", doi = "10.1021/cb500787b", issn = "1554-8929", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-131444540", note = "© 2015 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: September 30, 2014. Accepted: January 5, 2015. Published: January 5, 2015. \n\nWe thank J. Jackson and C. Dupre for assistance with the brain slices and J. C. Tapia, M. C. Wang, Z. Chen, D. Peterka, and R. Yuste for helpful discussions. We are grateful to Y. Shin for technical assistance with the in vivo mice experiments. W.M. acknowledges support from Columbia University, an National Institutes of Health Director’s New Innovator Award, the U.S. Army Research Office (W911NF-12-1-0594), the Brain Research Foundation, and an Alfred P. Sloan Research Fellowship. \n\nAuthor Contributions: L.W., Y.S., F.X., F.H., J.K.H., and K.L.T. performed experiments and analyzed data. L.W., Y.S., and W.M. designed the experiments. L.W. and W.M. conceived the concept and wrote the article. \n\nThe authors declare the following competing financial interest(s): Columbia University has filed a patent application based on this work.", revision_no = "21", abstract = "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. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86931, title ="Vibrational Imaging of Glucose Uptake in Live Cells and Tissues by Stimulated Raman Scattering Microscopy", author = "Hu, Fanghao and Chen, Zhixing", journal = "Biophysical Journal", volume = "108", number = "2", pages = "480A", month = "January", year = "2015", doi = "10.1016/j.bpj.2014.11.2621", issn = "0006-3495", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-135537234", note = "© 2015 Biophysical Society. Published by Elsevier Inc. \n\nAvailable online 27 January 2015. \n\nMeeting Abstract: 2420-Pos B557.", revision_no = "13", abstract = "Glucose is a ubiquitous energy source for virtually all living organisms. Its uptake activity closely reflects the cellular metabolic status in various physiological and pathological conditions. Extensive efforts such as positron emission tomography (PET), magnetic resonance imaging (MRI) and fluorescence microscopy have been made to image glucose uptake but all with technical limitations. Here, we report a novel vibrational microscopy platform to visualize glucose uptake in living cells and tissues with subcellular resolution and minimal perturbation by performing stimulated Raman scattering (SRS) on a new glucose analogue. Cancer cells with differing metabolic characteristics can be distinguished. Moreover, heterogeneous glucose uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues as well as in neuronal culture and mouse brain tissues. Therefore, by offering the distinct advantage of optical resolution yet without the undesirable influence of bulky fluorophores, SRS imaging of glucose uptake will be a valuable tool to study energy demands of living systems, particularly in tumors and brain.", } @thesis {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86925, title ="Nonlinear optical microscopy for the invisible: vibrational imaging of small molecules in live cells and electronic imaging of fluorophores into the ultra deep", author = "Wei, Lu", month = "January", year = "2015", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-131246246", revision_no = "11", abstract = "Nonlinear optical microscopy (NOM) has become increasingly popular in biomedical research in recent years with the developments of laser sources, contrast mechanisms, novel probes and etc. One of the advantages of NOM over the linear counterpart is the ability to image deep into scattering tissues or even on the whole animals. This is due to the adoption of near-infrared excitation that is of less scattering than visible excitation, and the intrinsic optical sectioning capability minimizing the excitation background beyond focal volume. Such an advantage is particularly prominent in two-photon fluorescence microscopy compared to one-photon fluorescence microscopy. In addition, NOM may provide extra molecular information (e.g. second harmonic generation and third harmonic generation) or stronger signal (e.g. stimulated Raman scattering and coherent anti-Stokes Raman scattering compared to spontaneous Raman scattering), because of the nonlinear interaction between strong optical fields and molecules. However, the merits of NOM are not yet fully exploited to tackle important questions in biomedical research. This thesis contributes to the developments of NOM in two aspects that correspond to two fundamental problems in biomedical imaging: first, how to non invasively image small functional biomolecules in live biological systems (Chapters 1-4); second, how to extend the optical imaging depth inside scattering tissues (Chapters 5-6). The ability to non-perturbatively image vital small biomolecules is crucial for understanding the complex functions of biological systems. However, it has proven to be highly challenging with the prevailing method of fluorescence microscopy. Because it requires the utilization of large-size fluorophore tagging (e.g., the Green Fluorescent Protein tagging) that would severely perturb the natural functions of small bio-molecules. Hence, we devise and construct a nonlinear Raman imaging platform, with the coupling of the emerging stimulated Raman scattering (SRS) microscopy and tiny vibrational tags, which provides superb sensitivity, specificity and biocompatibility for imaging small biomolecules (Chapters 1-4). Chapter 1 outlines the theoretical background for Raman scattering. Chapter 2 describes the instrumentation for SRS microscopy, followed with an overview of recent technical developments. Chapter 3 depicts the coupling of SRS microscopy with small alkyne tags (C≡C) to sensitively and specifically image a broad spectrum of small and functionally vital biomolecules (i.e. nucleic acids, amino acids, choline, fatty acids and small molecule drugs) in live cells, tissues and animals. Chapter 4 reports the combination of SRS microscopy with small carbon-deuterium (C-D) bonds to probe the complex and dynamic protein metabolism, including protein synthesis, degradation and trafficking, with subcellular resolution through metabolic labeling. It is to my belief that the coupling of SRS microscopy with alkyne or C-D tags will be readily applied in answering key biological questions in the near future. The remaining chapters of this thesis (Chapters 5-6) present the super-nonlinear fluorescence microscopy (SNFM) techniques for extending the optical imaging depth into scattering tissues. Unlike SRS microscopy that is an emerging technique, multiphoton microscopy (mainly referred as two-photon fluorescence microscopy), has matured over 20 years with its setup scheme and biological applications. Although it offers the deepest penetration in the optical microscopy, it still poses a fundamental depth limit set by the signal-to-background ratio when imaging into scattering tissues. Three SNFM techniques are proposed to extend such a depth limit: unlike the conventional multiphoton microscopy whose nonlinearity stems from virtual-states mediated simultaneous interactions between the incident photons and the molecules, the high-order nonlinearity of the SNFM techniques that we have conceived is generated through real-state mediated population-transfer kinetics. In particular, Chapter 5 demonstrates the multiphoton activation and imaging (MPAI) microscopy, which adopts a new class of fluorophores, the photoactivatable fluorophores, to significantly extend the fundamental imaging depth limit. Chapter 6 theoretically and analytically depicts two additional SNFM techniques of stimulated emission reduced fluorescence (SERF) microscopy and focal saturation microscopy. Both MPAI and focal saturation microscopies exhibit a fourth order power dependence, which is effectively a four-photon process. SERF presents a third order power dependence for a three-photon process.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91011, title ="Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette", author = "Chen, Zhixing and Paley, Daniel W.", journal = "Journal of the American Chemical Society", volume = "136", number = "22", pages = "8027-8033", month = "June", year = "2014", doi = "10.1021/ja502706q", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039582", note = "© 2014 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: March 17, 2014. Published: May 21, 2014. \n\nWe thank F. Hu, Y. Shen, and M. Jimenez for helpful discussions. D.W.P. and C.N. acknowledge support from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, U.S. Department of Energy (DOE) under award number DE-FG02-01ER15264. W.M. acknowledges support from NIH Director’s New Innovator Award and Alfred P. Sloan Research Fellowship. \n\nThe authors declare the following competing financial interest(s): Z.C., L.W., and W.M. are the inventors of a patent application filed by Columbia University. R.A.F. has an interest in Schrodinger, Inc. R.A.F. has a significant financial stake in Schrödinger, Inc., is a consultant to Schrödinger, Inc. and is on the Scientific Advisory Board of Schrödinger, Inc.", revision_no = "17", abstract = "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 ^(13)C-based isotopic editing strategy. We first synthesized ^(13)C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-^(13)C (^(13)C≡^(12)C) and bis-^(13)C (^(13)C≡^(13)C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled (^(12)C≡^(12)C) 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.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86927, title ="Live-cell quantitative imaging of proteome degradation by stimulated Raman scattering", author = "Shen, Yihui and Xu, Fang", journal = "Angewandte Chemie International Edition", volume = "53", number = "22", pages = "5596-5599", month = "May", year = "2014", doi = "10.1002/anie.201310725", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-133129304", note = "© 2014 WILEY‐VCH. \n\nReceived: December 10, 2013. Published online: April 15, 2014. \n\nW.M. acknowledges support from National Institutes of Health Director’s New Innovator Award and Sloan Research Fellowship.", revision_no = "15", 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 ^(13)C-phenylalanine is used to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous ^(12)C-phenylalanine and incorporated ^(13)C-phenylalanine as protein markers for the original and nascent proteomes, respectively, and the decay of the former wasquantified through ^(12)C/(^(12)C + ^(13)C) 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.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91012, title ="Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling", author = "Hu, Fanghao and Wei, Lu", journal = "Analyst", volume = "139", number = "10", pages = "2312-2317", month = "May", year = "2014", doi = "10.1039/c3an02281a", issn = "0003-2654", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039701", note = "© Royal Society of Chemistry 2014. \n\nReceived 11th December 2013, Accepted 18th January 2014, First published on 20th January 2014. \n\nWe thank M. Chalfie, Z. Chen and L. Zhang for stimulating discussion and Dr Y. Shin for providing mouse hippocampal neurons. W. Min acknowledges support from NIH Director's New Innovator Award. \n\nThe authors declare no competing financial interest.", revision_no = "14", abstract = "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.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86929, title ="Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering", author = "Wei, Lu and Hu, Fanghao", journal = "Nature Methods", volume = "11", number = "4", pages = "410-412", month = "April", year = "2014", doi = "10.1038/nmeth.2878", issn = "1548-7091", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-133705699", note = "© 2014 Macmillan Publishers Limited. \n\nReceived 2 October 2013; accepted 29 January 2014; published online 2 March 2014. \n\nWe thank L. Zhang, L. Brus, V.W. Cornish, D. Peterka and R. Yuste for helpful discussions. We are grateful to Y. Shin and X. Gao for technical assistance. W.M. acknowledges support from Columbia University, a US National Institutes of Health Director's New Innovator Award, the US Army Research Office (W911NF-12-1-0594) and an Alfred P. Sloan Research Fellowship. \n\nAuthor Contributions: L.W., F.H., Y.S., Z.C., Y.Y., C.-C.L. and M.C.W. performed experiments and analyzed data. L.W. and W.M. conceived the concept, designed the experiments and wrote the paper. \n\nCompeting interests: Columbia University, which L.W., F.H., Y.S., Z.C. and W.M. are affiliated with, has filed a patent application based on this work.", revision_no = "15", abstract = "Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors. ", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86928, title ="Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging", author = "Wei, Lu and Zhu, Xinxin", number = "8948", pages = "Art. No. 894825", month = "February", year = "2014", doi = "10.1117/12.2038753", isbn = "9780819498618", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180608-133651608", note = "© 2014 Society of Photo-Optical Instrumentation Engineers (SPIE).", revision_no = "11", abstract = "Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91013, title ="Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy", author = "Wei, Lu and Yu, Yong", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "110", number = "28", pages = "11226-11231", month = "July", year = "2013", doi = "10.1073/pnas.1303768110", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039803", note = "© 2013 National Academy of Sciences. \n\nEdited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved May 31, 2013 (received for review February 27, 2013) \n\nWe thank F. Hu, Z. Chen, V. W. Cornish, D. Peterka, and R. Yuste for helpful discussion. We are grateful to S. Buffington, M. Costa-Mattioli, and M. Sakamoto for providing hippocampal neurons, and Y. Li for his assistance on the spontaneous Raman microscope. We acknowledge support from Ellison Medical Foundation fellowships (to M.C.W.) and National Institutes of Health Director’s New Innovator Award (to W.M.). \n\nAuthor contributions: L.W., M.C.W., and W.M. designed research; L.W., Y.Y., and Y.S. performed research; L.W. analyzed data; and L.W., Y.Y., Y.S., M.C.W., and W.M. wrote the paper. \n\nConflict of interest statement: Columbia University has filed a patent application based on this work. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303768110/-/DCSupplemental.", revision_no = "11", abstract = "Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91015, title ="What can stimulated emission do for bioimaging?", author = "Wei, Lu and Min, Wei", journal = "Annals of the New York Academy of Sciences", volume = "1293", pages = "1-7", month = "July", year = "2013", doi = "10.1111/nyas.12079", issn = "0077-8923", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040019", note = "© 2013 New York Academy of Sciences.\n\nThe authors thank Zhixing Chen and Rafael Yuste for helpful discussions. W.M. acknowledges the start‐up funds from Columbia University, and grant support from the Kavli Institute for Brain Science. \n\nThe authors declare no conflicts of interest. \n\nIssue: Blavatnik Awards for Young Scientists 2012.", revision_no = "11", abstract = "Advances in bioimaging have revolutionized our ability to study life phenomena at a microscopic scale. In particular, the stimulated emission process, a universal mechanism that competes with spontaneous emission, has emerged as a powerful driving force for advancing light microscopy. The present review summarizes and compares three related techniques that each measure a different physical quantity involved in the stimulated emission process in order to tackle various challenges in light microscopy. Stimulated emission depletion microscopy, which detects the residual fluorescence after quenching, can break the diffraction‐limited resolution barrier in fluorescence microscopy. Stimulated emission microscopy is capable of imaging nonfluorescent but absorbing chromophores by detecting the intensity gain of the stimulated emission beam. Very recently, stimulated emission reduced fluorescence microscopy has been proposed, in which the reduced fluorescence due to focal stimulation is measured to extend the fundamental imaging‐depth limit of two‐photon microscopy. Thus, through ingenious spectroscopy design in distinct microscopy contexts, stimulated emission has opened up several new territories for bioimaging, allowing examination of biological structures that are ever smaller, darker, and deeper.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91014, title ="Frustrated FRET for high-contrast high-resolution two-photon imaging", author = "Xu, Fang and Wei, Lu", journal = "Optics Express", volume = "21", number = "12", pages = "14097-14108", month = "June", year = "2013", doi = "10.1364/oe.21.014097", issn = "1094-4087", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039911", note = "© 2013 Optical Society of America. \n\nReceived 20 Feb 2013; revised 1 Apr 2013; accepted 2 Apr 2013; published 5 Jun 2013. \n\nWe thank L. Zhang, X. Zhu, L. Brus, R. Yuste, D. Peterka, V. Cornish and M. Jimenez for helpful discussions. W.M. acknowledges support from Kavli Institute for Brain Science and RISE program of Columbia University.", revision_no = "9", abstract = "Two-photon fluorescence microscopy has become increasingly popular in biomedical research as it allows high-resolution imaging of thick biological specimen with superior contrast and penetration than confocal microscopy. However, two-photon microscopy still faces two fundamental limitations: 1) image-contrast deterioration with imaging depth due to out-of-focus background and 2) diffraction-limited spatial resolution. Herein we propose to create and detect high-order (more than quadratic) nonlinear signals by harnessing the frustrated fluorescence resonance energy transfer (FRET) effect within a specially designed donor-acceptor probe pair. Two distinct techniques are described. In the first method, donor fluorescence generated by a two-photon laser at the focus is preferentially switched on and off by a modulated and focused one-photon laser beam that is able to block FRET via direct acceptor excitation. The resulting image, constructed from the enhanced donor fluorescence signal, turns out to be an overall three-photon process. In the second method, a two-photon laser at a proper wavelength is capable of simultaneously exciting both the donor and the acceptor. By sinusoidally modulating the two-photon excitation laser at a fundamental frequency ω, an overall four-photon signal can be isolated by demodulating the donor fluorescence at the third harmonic frequency 3ω. We show that both the image contrast and the spatial resolution of the standard two-photon fluorescence microscopy can be substantially improved by virtue of the high-order nonlinearity. This frustrated FRET approach represents a strategy that is based on extracting the inherent nonlinear photophysical response of the specially designed imaging probes.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91017, title ="Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag", author = "Gatzogiannis, Evangelos and Chen, Zhixing", journal = "Chemical Communications", volume = "48", number = "69", pages = "8694-8696", month = "September", year = "2012", doi = "10.1039/c2cc33133k", issn = "1359-7345", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040204", note = "© Royal Society of Chemistry 2012. \n\nReceived 1st May 2012, Accepted 6th July 2012. \n\nThis work was supported by the National Institutes of Health (U54 GM087519 and RC1GM091804 to V. W. C) and by the start up funds from Columbia University (to W. M.). We thank Dr Steffen Jockusch for experimental assistance. \n\nV. W. C. holds patents on the TMP-tag technology, and the technology is licensed and commercialized by Active Motif.", revision_no = "14", abstract = "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.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91018, title ="Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores", author = "Chen, Zhixing and Wei, Lu", journal = "Optics Express", volume = "20", number = "17", pages = "18525-18536", month = "August", year = "2012", doi = "10.1364/oe.20.018525", issn = "1094-4087", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040297", note = "© 2012 Optical Society of America. \n\nReceived 12 Apr 2012; revised 18 Jun 2012; accepted 19 Jun 2012; published 30 Jul 2012. \n\nZhixing Chen, Lu Wei and Xinxin Zhu contributed equally to this work. We thank Ya-Ting Kao, Fang Xu, Louis Brus, Rafael Yuste, Nicholas Turro, Virginia Cornish, Darcy Peterka, Christophe Dupre and Miguel Jimenez for helpful discussions. We are grateful to Virginia Cornish for sharing lab equipments and Keith Yeager for assistance on Leica microscope. W.M. acknowledges the startup funds from Columbia University, and grant support from Kavli Institute for Brain Science.", revision_no = "10", abstract = "It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91019, title ="Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging", author = "Wei, Lu and Chen, Zhixing", journal = "Biomedical Optics Express", volume = "3", number = "6", pages = "1465-1475", month = "June", year = "2012", doi = "10.1364/boe.3.001465", issn = "2156-7085", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040395", note = "© 2012 Optical Society of America. \n\nReceived 27 Apr 2012; revised 19 May 2012; accepted 19 May 2012; published 22 May 2012. \n\nWe thank Ya-Ting Kao, Xinxin Zhu, Louis Brus, Rafael Yuste, Darcy Peterka, Virginia Cornish, Christophe Dupre and Miguel Jimenez for helpful discussions. W. M. acknowledges the startup funds from Columbia University, and grant support from Kavli Institute for Brain Science.", revision_no = "9", abstract = "Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/91016, title ="Pump-probe optical microscopy for imaging nonfluorescent chromophores", author = "Wei, Lu and Min, Wei", journal = "Analytical and Bioanalytical Chemistry", volume = "403", number = "8", pages = "2197-2202", month = "June", year = "2012", doi = "10.1007/s00216-012-5890-1", issn = "1618-2642", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040116", note = "© Springer-Verlag 2012. \n\nReceived: 5 December 2011 /Revised: 16 February 2012 / Accepted: 20 February 2012 / Published online: 13 March 2012. \n\nWe acknowledge discussions with X. S. Xie, C. W. Freudiger, S. Lu, S. Chong, B. G. Saar, G. R. Holtom, M. Roeffaers, D. Fu, X. Zhang, and R. Roy. \n\nPublished in the special issue Young Investigators in Analytical and Bioanalytical Science with Guest Editors S. Daunert, J. Bettmer, T. Hasegawa, Q. Wang and Y. Wei.", revision_no = "11", abstract = "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.", }