Converting lateral scanning into axial focusing to speed up three-dimensional microscopy

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.

Scalable integration of nano-,and microfluidics with hybrid two-photon lithography

Nanofluidic devices have great potential for applications in areas ranging from renewable energy to human health.A crucial requirement for the successful operation of nanofluidic devices is the ability to interface them in a scalable manner with the outside world.Here,we demonstrate a hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices.Using this approach we demonstrate the fabrication of molds from SU-8 photoresist with nanofluidic features down to 230 nm lateral width and channel heights from micron to sub-100 nm.Scanning electron microscopy and atomic force microscopy were used to characterize the printing capabilities of the system and show the integration of nanofluidic channels into an existing microfluidic chip design.The functionality of the devices was demonstrated through super-resolution microscopy,allowing the observation of features below the diffraction limit of light produced using our approach.Single molecule localization of diffusing dye molecules verified the successful imprint of nanochannels and the spatial confinement of molecules to 200 nm across the nanochannel molded from the master wafer.This approach integrates readily with current microfluidic fabrication methods and allows the combination of microfluidic devices with locally two-photon-written nano-sized functionalities,enabling rapid nanofluidic device fabrication and enhancement of existing microfluidic device architectures with nanofluidic features.