Among optical imaging techniques light sheet fluorescence microscopy is one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However light sheet microscopes are limited by volume scanning rate and/or camera speed. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. Our fast scan system supports 40 Hz imaging of 700 μm-thick volumes if camera speed is sufficient. We also address the camera speed limitation by introducing Distributed Planar Imaging (DPI), a scaleable technique that parallelizes image acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact, removable when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.

Among optical imaging techniques light sheet fluorescence microscopy stands out as one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However current-generation light sheet microscopes are limited by volume scanning rate and/or camera frame rate. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. We increase volume scanning rate to 40 Hz for volumes up to 700 µm thick and introduce Multi-Camera Image Sharing (MCIS), a technique to scale imaging rate by parallelizing acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact that can be removed by filtering when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.Improvements in calcium 1 and voltage 2,3 indicators have opened up the possibility of using fluorescence microscopy to observe interactions between neurons in the brain. Because neuronal computations occur on short timescales and are distributed over large tissue volumes, capturing these three-dimensional dynamics presents a major acquisition challenge. Motivated by this goal, 1 many groups have developed or refined microscopy techniques that increase the scale and/or speed of fluorescence microscopy 4-24 . These fast techniques can be broadly categorized by the way that they collect pixel information to form an image: point-scanning (confocal and two-photon) methods collect one or a few 16 pixels at a time, while line-scanning and widefield methods parallelize acquisition over hundreds to millions of pixels on a camera sensor.Fundamental physics-specifically, limits on the relaxation time of fluorophores, leading to a phenomenon known as fluorophore saturation 25 -dictates that single point-scanning imaging techniques have lesser potential speed than techniques that image many points in parallel. Traditional widefield imaging methods such as epifluorescence excel at this parallelization but pay a cost in axial resolution: they produce 2-dimensional images containing a mixture of fluorescent sources from multiple depths in the sample.Recently several approaches have been developed to separate signals by depth-referred to as optical sectioning-while imaging many points in parallel. Light-sheet fluorescence microscopy (LSFM) is one of the most popular of these approaches. Conventional LSFM performs optical sectioning by way of specialized hardware that limits excitation light to a single plane at a time while capturing an image of that plane with a camera. Thus the illuminated region is coincident with the image plane, allowing the entire image to be captured in parallel, while also avoiding unnecessary light exposure and affording LSFM with exceptionally ...

The design of ‘hunter’ viruses aimed at destroying human immunodeficiency virus (HIV) infected cells is an active area of research that has produced promising results in vitro. Hunters are designed to target exposed viral envelope proteins in the membranes of infected cells, but there is evidence that the hunter may also target envelope proteins of free HIV, inducing virus-virus fusion. In order to predict the effects of this fusion on therapy outcomes and determine whether fusion ability is advantageous for hunter virus design, we have constructed a model to account for the possibility of hunter-HIV fusion. The study was based on a target cell-limited model of HIV infection and it examined the hunter therapeutic effect on recovering the HIV main target cells, the activated CD4+ T lymphocytes. These cells assist in setting up an immune response to opportunistic infections. The study analyzed the hunter dual mechanisms to control infection and because of diverse estimates for viral production and clearance of HIV, simulations were examined at rates spanning an order of magnitude. Results indicate that without hunter-HIV fusion ability, hunters that kill HIV-infected cells lead to a substantial recovery of healthy cell population at both low and high HIV turnover rates. When hunter-HIV fusion is included, cell recovery was particularly enhanced at lower HIV turnover rates. This study shows that the fusion ability, in addition to hunter infection ability, could be a favorable attribute for improving the efficacy of hunter-viral therapy. These results provide support for the potential use of engineered viruses to control HIV and other viral infections.