ASLM

Two light-sheet microscopy techniques successfully decouple the trade-off between axial resolution and field of view: multiview deconvolution and ASLM. In the former, the sample is imaged from multiple directions (via sample rotation or by reversing the illumination and detection paths) and the complementary views computationally fused to create a high-resolution, large field of view image. However, this increases the illumination burden on the specimen, increases the data overhead, and requires deconvolution which is incompatible with biosensor-based imaging. In contrast, ASLM, a technology invented by Dean and Fiolka in 2015, uses aberration-free remote focusing to sweep a tightly focused Gaussian beam through the sample in its propagation direction synchronously with the rolling shutter of a CMOS camera. Here, the axial position of the illumination beam is deterministically controlled by translating the position of a piezo or voice coil mounted mirror that is optically conjugate to the specimen at the focal plane of a remote focusing objective. More recently, to accelerate the image acquisition rate of ASLM to 200 frames per second, we developed a novel method that converted a lateral galvanometer sweep into an aberration-free scan of the illumination beam. Importantly, ASLM does not require any computational post-processing, and delivers a large field of view (100 x 100 x 100µm) and the highest axial resolution (~400 nm) in LSFM in the absence of deconvolution or super-resolution techniques (e.g., structured illumination, stimulated emission, or localization). Because ASLM has isotropic resolution, it is routinely combined with advanced computer vision software, and has already provided tremendous insight into diverse biological phenomena. For example, when combined with the software package u-shape3D, ASLM was able to evaluate how Kras and PIP2 influence cell morphology and motion in 3D collagen environments. Importantly, because it uses refractive rather than diffractive optics, ASLM is entirely compatible with multiplexed excitation and detection of multiple biosensors and/or optogenetic probes. As such, ASLM is ideally suited to evaluate the molecular mechanisms underlying cell-cell interactions and metastatic dissemination in 3D extracellular matrix environments.