annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a eonfoeal microscope. It simultaneously detects far-, on-, and near-fo...annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a eonfoeal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the eonfoeal microscope into three sub-paths （signals）. Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confoeal microscope and its lateral resolution can be better than 0.2 um for A = 632.8 nm, the numerical aperture of the lens of the microscope is NA = 0.85, and the normalized radius e = 0.5.展开更多
Cells are crowded microenvironments filled with macromolecules undergoing constant phys- ical and chemical interactions. The physicochemical makeup of the cells aff）cts various cellular responses, determines cell-cel...Cells are crowded microenvironments filled with macromolecules undergoing constant phys- ical and chemical interactions. The physicochemical makeup of the cells aff）cts various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties diff）r between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process terrned signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies light. We summarize recent work which uses light to both visualize the cellular environment and also control intracel- lular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends un- precedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.展开更多
The resolution of conventional optical microscopy is only -200 nm, which is becoming less and less sufficient for a variety of applications. In order to surpass the diffraction limited resolution, super-resolution mic...The resolution of conventional optical microscopy is only -200 nm, which is becoming less and less sufficient for a variety of applications. In order to surpass the diffraction limited resolution, super-resolution microscopy （SRM） has been developed to achieve a high resolution of one to tens of nanometers. The techniques involved in SRM can be assigned into two broad categories, namely ＂true＂ super-resolution techniques and ＂functional＂ super-resolution techniques. In ＂functional＂ super-resolution techniques, stochastic super-resolution microscopy （SSRM） is widely used due to its low expense, simple operation, and high resolution. The principle process in SSRM is to accumulate the coordinates of many diffraction-limited emitters （e.g., single fluorescent molecules） on the object by localizing the centroids of the point spread functions （PSF）, and then reconstruct the image of the object using these coordinates. When the diffraction-limited emitters take part in a catalytic reaction, the activity distribution and kinetic information about the catalysis by nanoparticles can be obtained by SSRM. SSRM has been applied and exhibited outstanding advantages in several fields of catalysis, such as metal nanoparticle catalysis, molecular sieve catalysis, and photocatalysis. Since SSRM is able to resolve the catalytic activity within one nanoparticle, it promises to accelerate the development and discovery of new and better catalysts. This review will present a brief introduction to SRM, and a detailed description of SSRM and its applications in nano-catalysis.展开更多
Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy w...Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.展开更多
Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluoroph...Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-guest compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.展开更多
基金Project supported by the National Natural Science Foundation of China （Grant No 50475035）, the Doctoral Program of Higher Education of China （Grant No 20050213035） and the Program for New Century Excellent Talents in University of China （Grant No NCET-05-0348）.
文摘annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a eonfoeal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the eonfoeal microscope into three sub-paths （signals）. Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confoeal microscope and its lateral resolution can be better than 0.2 um for A = 632.8 nm, the numerical aperture of the lens of the microscope is NA = 0.85, and the normalized radius e = 0.5.
基金supported by the School of Molecular Cell Biology at the University of Illinois at Urbana-Champaign
文摘Cells are crowded microenvironments filled with macromolecules undergoing constant phys- ical and chemical interactions. The physicochemical makeup of the cells aff）cts various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties diff）r between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process terrned signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies light. We summarize recent work which uses light to both visualize the cellular environment and also control intracel- lular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends un- precedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.
文摘The resolution of conventional optical microscopy is only -200 nm, which is becoming less and less sufficient for a variety of applications. In order to surpass the diffraction limited resolution, super-resolution microscopy （SRM） has been developed to achieve a high resolution of one to tens of nanometers. The techniques involved in SRM can be assigned into two broad categories, namely ＂true＂ super-resolution techniques and ＂functional＂ super-resolution techniques. In ＂functional＂ super-resolution techniques, stochastic super-resolution microscopy （SSRM） is widely used due to its low expense, simple operation, and high resolution. The principle process in SSRM is to accumulate the coordinates of many diffraction-limited emitters （e.g., single fluorescent molecules） on the object by localizing the centroids of the point spread functions （PSF）, and then reconstruct the image of the object using these coordinates. When the diffraction-limited emitters take part in a catalytic reaction, the activity distribution and kinetic information about the catalysis by nanoparticles can be obtained by SSRM. SSRM has been applied and exhibited outstanding advantages in several fields of catalysis, such as metal nanoparticle catalysis, molecular sieve catalysis, and photocatalysis. Since SSRM is able to resolve the catalytic activity within one nanoparticle, it promises to accelerate the development and discovery of new and better catalysts. This review will present a brief introduction to SRM, and a detailed description of SSRM and its applications in nano-catalysis.
文摘Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional （3D） localisation microscopy by engineering of the point-spread function （PSF） of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions （standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively）. The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.
基金supported by the National Natural Science Foundation of China(31330082,21373200,21525314)the Instrument Developing Project of the Chinese Academy of Sciences(YZ201455)
文摘Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-guest compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.
