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
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.
