Person: Zhang, Xu
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Zhang
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Xu
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Zhang, Xu
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Publication Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy(American Chemical Society, 2012) Fu, Dan; Lu, Fake; Zhang, Xu; Freudiger, Christian Wilhelm; Pernik, Douglas R.; Holtom, Gary; Xie, XiaoliangStimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.Publication Stimulated Raman Scattering Imaging of Biomolecules and Single Cell Transcriptome Analysis of Mouse Retina(2015-05-19) Zhang, Xu; Xie, Xiaoliang S.; Weitz, David A.; Sanes, Joshua R.; Loncar, MarkoComplex information within biological systems is being uncovered at an unprecedented speed thanks to the rapid technical development of a wide variety of research tools, among which imaging and sequencing technologies are attracting big attention in recent years. Optical imaging enables the visualization of the spatial distribution of biomolecules at cellular level, allowing deeper understanding of the structure and dynamics of biological systems. Fluorescence microscopy has contributed greatly to our understanding of these processes, but it relies on the use of fluorescent labels or dyes. These labels may perturb the studied systems especially for imaging small molecules, and the photobleaching problem also limits the long-term biological dynamics observation within living cells. In the first part of this dissertation, we introduce the recent development of Stimulated Raman scattering (SRS) microscopy as a noninvasive imaging technique with superior sensitivity, molecular specificity at video-rate imaging speed. It has superseded coherent anti-Stokes Raman scattering (CARS) microscopy due to the absence of non-resonant background and automatic phase matching. However, SRS imaging has been mostly demonstrated for the visualization of lipid and protein with long vibrational wavenumbers. We extend the detectability of SRS imaging into the crowded fingerprint region with characteristic signatures of more biomolecules such as nucleic acids in live cells (Chapter 2), unsaturated lipid and aromatic amino acid in multiphasic food products (Chapter 3). Noninvasiveness of SRS imaging also brings new opportunities to biomedical applications and we demonstrate its feasibility as a potential pathology diagnostic tool by generating comparable image contrast as golden standard H&E staining in human brain frozen sections (Chapter 4). We further extend SRS imaging to real-time multiband detection using a novel modulation multiplex approach (Chapter 5). The rapid development of high throughput sequencing technologies has enabled whole genome and transcriptome wide analysis at faster speed and affordable cost, but a large number of cells are often still required for these analyses. However, cell-to-cell variation is significant and may carry important indication to the study of complex wiring in the nervous systems. In this second part of the dissertation, we explore the heterogeneity of retina using a recently developed single cell transcriptome amplification technique based on Multiple Annealing Looping Based Amplification Cycles (MALBAC), which is superior to other single cell techniques with its low amplification bias, high reproducibility rate and low dropout rate. We first classify different retinal cell populations (photoreceptor cells vs. retinal ganglion cells) and closely related subpopulations (different direction selective retinal ganglion cells) (Chapter6). We further study the molecular divergence of an unsolved ON-OFF retina circuit responsible for direction selectivity function. We show that the inhibitory interneurons responsible for this function can be classified into two clusters based on the single cell transcriptome data. This clustering result strongly correlates with the ON-OFF starburst amacrine cells (SACs) based on the immunostaining results of the identified differential genes. The newly reported differential genes can potentially be used as molecular markers for ON-OFF SACs with more validation underway (Chapter 7). These new findings open up more opportunities for the functional studies on the direction-selective circuit in retina.Publication Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy(John Wiley and Sons, 2012) Zhang, Xu; Roeffaers, Maarten B. J.; Basu, Srinjan; Daniele, Joseph R.; Fu, Dan; Freudiger, Christian Wilhelm; Holtom, Gary R.; Xie, XiaoliangImaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.