In optical microscopy,the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging.Recently,by conjugating either a movable mirror to the image plane in a remot...In optical microscopy,the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging.Recently,by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens(ETL)to the back focal plane,rapid axial scanning has been achieved.However,mechanical actuation of a mirror limits the axial scanning rate(usually only 10–100 Hz for piezoelectric or voice coil-based actuators),while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging.In an effort to overcome these limitations,we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging.Using a galvanometric mirror,we scan a laser beam laterally in a remote-focusing arm,which is then back-reflected from different heights of a mirror in the image space.We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude,allowing the quantification of rapid vesicular dynamics in three dimensions.We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope,which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.展开更多
基金funded by grants from the Cancer Prevention Research Institute of Texas(RR160057 to R.F.)the National Institutes of Health(F32GM117793 to K.M.D.and R33CA235254 and R35GM133522 to R.F.)+2 种基金funding from the UK Engineering and Physical Sciences Research Council,EPSRC(grants EP/L015889/1 and EP/H018301/1)the Wellcome Trust(grants 3-3249/Z/16/Z and 089703/Z/09/Z)the UK Medical Research Council,MRC(grants MR/K015850/1 and MR/K02292X/1),MedImmune,and Infinitus(China)Ltd.
文摘In optical microscopy,the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging.Recently,by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens(ETL)to the back focal plane,rapid axial scanning has been achieved.However,mechanical actuation of a mirror limits the axial scanning rate(usually only 10–100 Hz for piezoelectric or voice coil-based actuators),while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging.In an effort to overcome these limitations,we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging.Using a galvanometric mirror,we scan a laser beam laterally in a remote-focusing arm,which is then back-reflected from different heights of a mirror in the image space.We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude,allowing the quantification of rapid vesicular dynamics in three dimensions.We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope,which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.
