Hyperspectral stimulated Raman scattering(SRS)microscopy is rapidly becoming an established method for chemical and biomedical imaging due to the combination of high spatial resolution and chemical information contain...Hyperspectral stimulated Raman scattering(SRS)microscopy is rapidly becoming an established method for chemical and biomedical imaging due to the combination of high spatial resolution and chemical information contained within the three-dimensional data set.Chemometric analysis techniques based on linear unmixing,or multivariate analysis,have become indispensable when visualizing hyperspectral data sets.The application of spectral phasor analysis has also been extremely fruitful in this regard,providing a convenient method to retrieve the spatial and chemical components of the data set.Here,we demonstrate the application of spectral phasor analysis for unmixing the overlapping spectral features within the cell-silent region of the SRS spectrum(2000−2300 cm^(−1)).In doing so,we show it is possible to identify specific Raman signals for DNA,proteins,and lipids following glucose-d7 metabolism in dividing cells.In addition,we show that spectral phasor analysis is capable of distinguishing different bioorthogonal Raman signals including alkynes and carbon−deuterium(C−D)bonds.We demonstrate the application of spectral phasor analysis for multicomponent unmixing of bioorthogonal Raman groups for high-content cellular imaging applications.展开更多
Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains chal...Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.展开更多
基金University of Strathclyde and the EPSRC(EP/N010914/1)for funding.
文摘Hyperspectral stimulated Raman scattering(SRS)microscopy is rapidly becoming an established method for chemical and biomedical imaging due to the combination of high spatial resolution and chemical information contained within the three-dimensional data set.Chemometric analysis techniques based on linear unmixing,or multivariate analysis,have become indispensable when visualizing hyperspectral data sets.The application of spectral phasor analysis has also been extremely fruitful in this regard,providing a convenient method to retrieve the spatial and chemical components of the data set.Here,we demonstrate the application of spectral phasor analysis for unmixing the overlapping spectral features within the cell-silent region of the SRS spectrum(2000−2300 cm^(−1)).In doing so,we show it is possible to identify specific Raman signals for DNA,proteins,and lipids following glucose-d7 metabolism in dividing cells.In addition,we show that spectral phasor analysis is capable of distinguishing different bioorthogonal Raman signals including alkynes and carbon−deuterium(C−D)bonds.We demonstrate the application of spectral phasor analysis for multicomponent unmixing of bioorthogonal Raman groups for high-content cellular imaging applications.
基金supported by the following grants:National Natural Science Foundation of China(62125504,62361166631)STI 2030-Major Projects(2021ZD0200401)+1 种基金the Fundamental Research Funds for the Central Universities(226-2022-00201)the Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.
