INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secret...INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.展开更多
A spectrometric setup to perform total internal reflection fluorescence (TIRF) and synchronous TIRF measurements at solid/liquid interfaces is presented. The combination of TIRF and synchronous fluorescence was propo...A spectrometric setup to perform total internal reflection fluorescence (TIRF) and synchronous TIRF measurements at solid/liquid interfaces is presented. The combination of TIRF and synchronous fluorescence was proposed to analyze simultaneously different components at interfaces. The TIRF excitation, emission and synchronous spectra of a watersoluble porphyrin were obtained from water/glass interface using this setup without the existence of a surfactant.展开更多
Total internal reflection fluorescence spectroscopy (TIRF) and synchronous scanning technique were combined to study the adsorption behavior of the meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) at the glass-water...Total internal reflection fluorescence spectroscopy (TIRF) and synchronous scanning technique were combined to study the adsorption behavior of the meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) at the glass-water interface without any surfactant. The pH dependence of synchronous fluorescence signal at the interface was analyzed. Both unprotonated (TPPS4-) and diprotonated (H2TPPS2-) forms of TPPS were observed at the interface. But the interface favored the adsorption of. The apparent estimated pKa2 value shifted from 5.00 in the bulk solution to 2.7 at the interface. STIRF provides a good technique to study multi-component systems at the interface.展开更多
TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.I...TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.展开更多
Optical microscopy promises researchers to soe most tiny substances directly.However,the resolution of conventional microscopy is resticted by the diffraction limit.This makes it a challenge to observe subcellular pro...Optical microscopy promises researchers to soe most tiny substances directly.However,the resolution of conventional microscopy is resticted by the diffraction limit.This makes it a challenge to observe subcellular processes happened in nanoscale.The development of super-resolution microscopy provides a solution to this challenge.Here,we briefly review several commonly used super-resolution techniques,explicating their basic principles and applications in biological science,especially in neuroscience.In addition,characteristics and limitations of each techrique are compared to provide a guidance for biologists to choose the most suitable tool.展开更多
文摘INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.
文摘A spectrometric setup to perform total internal reflection fluorescence (TIRF) and synchronous TIRF measurements at solid/liquid interfaces is presented. The combination of TIRF and synchronous fluorescence was proposed to analyze simultaneously different components at interfaces. The TIRF excitation, emission and synchronous spectra of a watersoluble porphyrin were obtained from water/glass interface using this setup without the existence of a surfactant.
文摘Total internal reflection fluorescence spectroscopy (TIRF) and synchronous scanning technique were combined to study the adsorption behavior of the meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) at the glass-water interface without any surfactant. The pH dependence of synchronous fluorescence signal at the interface was analyzed. Both unprotonated (TPPS4-) and diprotonated (H2TPPS2-) forms of TPPS were observed at the interface. But the interface favored the adsorption of. The apparent estimated pKa2 value shifted from 5.00 in the bulk solution to 2.7 at the interface. STIRF provides a good technique to study multi-component systems at the interface.
基金Project supported by the National Natural Science Foundation ofChina (No. 30770596)the Key Laboratory for Biomedical En-gineering of Ministry of Education of China
文摘TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.
基金support from National Basic Research Program of China (973 Program) (2015CB352005)National Natural Science Foundation of China (No.6142780065,31571110,81527901)+1 种基金Natural Science Foundation of Zhejiang Province of China (No.Y16F050002)the Fundamental Research Funds for the Central Universities.
文摘Optical microscopy promises researchers to soe most tiny substances directly.However,the resolution of conventional microscopy is resticted by the diffraction limit.This makes it a challenge to observe subcellular processes happened in nanoscale.The development of super-resolution microscopy provides a solution to this challenge.Here,we briefly review several commonly used super-resolution techniques,explicating their basic principles and applications in biological science,especially in neuroscience.In addition,characteristics and limitations of each techrique are compared to provide a guidance for biologists to choose the most suitable tool.
