Spatiotemporal Multiplexing for Fast 3d Confocal Microscopy Using Acoustic Tunable Lenses

Fast optical imaging of three-dimensional (3D) specimens is pivotal for the understanding of fundamental processes in life sciences. In most cases, 3D imaging is performed by acquiring a stack of 2D sections each at a different focal position. Notably, contrast and 3Dreconstruction accuracy are maximized with decreasing the optical thickness of 2D sections. This has spurred the development of several techniques capable of optical sectioning including confocal or two-photon microscopy. In such cases, fast and high-resolution acquisition of 2D sections can be successfully accomplished. However, retrieving information from a 3D volume is based on an inherently slow stepwise process: after acquiring each 2D image, one must mechanically move the focus in the axial direction and wait for proper repositioning to occur. This need of mechanical z-focus control constitutes one of the main limitations for 3D fast imaging. Here, we show a rapid and single-step method for imaging an entire 3D volume in confocal microscopy without the need of mechanical movement [1]. Our approach is based on axially scanning the focus at frequencies of 143 kHz or above by using the refractive index gradients induced in a sound-driven liquid lens [2]. Such high scanning speed enables efficient spatiotemporal multiplexing of the excitation beam on a pixel by pixel basis (Figure 1). By using appropriate demultiplex readout, photons collected at a certain instant can be assigned to their corresponding focal plane. In this way, in a single XY scan that would usually result in the capture of a single 2D image, we can simultaneously acquire multiple sections and reconstruct an entire 3D image. We show imaging of different biological and calibration fluorescent samples and we compare these images with those obtained with traditional z-scanners. Based on these results, we find that image quality is close to that obtained with traditional confocal methods while acquisition speed is greatly improved being only limited by signal-tonoise-ratio.