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Research And Application Of Hyperspectral Stimulated Raman Scattering Microscopic Imaging Based On Molecular Probes

Posted on:2020-07-31Degree:MasterType:Thesis
Country:ChinaCandidate:C DingFull Text:PDF
GTID:2381330590476475Subject:Organic Chemistry
Abstract/Summary:PDF Full Text Request
The application of Raman scattering spectroscopy has made great progress in the last century.From the initial spontaneous Raman scattering spectroscopy to surface-enhanced Raman scattering spectroscopy,coherent anti-Stokes Raman scattering spectroscopy and stimulated Raman scattering?SRS?,each technological improvement has considerably expanded its application in biological imaging field.Stimulated Raman scattering microscopy has been developing as an emerging imaging technology in the last decade.The imaging contrast of this technique derives from the"fingerprint spectrum"produced by different chemical bonds in molecules during Raman scattering process.Therefore,it boasts of good selectivity and specificity for different molecules.In addition,compared with conventional SRS imaging technology,hyperspectral stimulated Raman scattering microscopy system using femtosecond laser as light source is of high resolution and detection sensitivity.This system generates a Raman spectrum at each imaging pixel,thus distinguishing different molecules with partially overlapping Raman bands and providing abundant chemical bond information.Therefore,the application of hyperspectral SRS micro-imaging technology has become a research focus in recent years.The development of SRS micro-imaging technology really does facilitate the understanding of the metabolic process.The chemical bonds of various macromolecules in the tissues and cells of organisms are of scarce disparity.Therefore,in the imaging process,the signal interference among various bio-molecules is intense.Consequently,the useful information from the obtained images is limited.To reduce or eliminate this interference,researchers synthesized a series of Raman probes with Raman signals located in Raman-silent region of cells(1800-2800 cm-1)for bio-imaging research.These molecules are mainly alkynyl modified or deuterium labeled nucleosides,amino acids,glucose or precursors of various lipid compounds.Combined with SRS micro-imaging technology,the synthesis and application of these probes have enriched people's understanding of the metabolic processes of nucleic acids,proteins,polysaccharides and lipids.In order to obtain images with higher resolution,researchers have been managing to synthesize novel Raman probe molecules with stronger vibration signals.Most of these molecules contain one polyyne structure,whose vibration spectral linewidth is about 1 nanometer,with Raman signal being strong as well as stable.What's more,the signal intensity of molecules is of good linear relationship with its concentration,so these probe molecules can also be used for quantitative or semi-quantitative imaging analysis of biological systems.In this paper,we synthesized a Raman probe-Lyso-BADY-from the structure of diaryl butadiyne with a morpholine moiety,which can target lysosomes in living cells.Raman spectrum showed that the Raman shift of the molecule was at 2250 cm-1,and the Raman signal intensity was 28 times of that of 5-ethynyl-2'-deoxyuridine?EdU?.The distribution and state of lysosomes in living cells were successfully observed by co-localization analysis with commercial dye Lyso-Tracker-Red under the hyperspectral SRS microscopy.For the next step,the photostability experiment showed that compared with two-photon fluorescence imaging images,hyperspectral SRS images did not fade even when cells were exposed to longer laser irradiation time,which indicated that Lyso-BADY molecules in living cells maintained strong Raman signals,and did not decompose after a long laser irradiation time.The results of this study enrich and expand the application of the emerging Raman probe molecules and hyperspectral SRS technology in organelle imaging,which will positively push forward the development of SRS micro-imaging technology in the bio-chemistry field.
Keywords/Search Tags:stimulated Raman scattering, hyperspectral, Raman-silent region of cells, Raman probe molecules, lysosomes, photostability
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