Fast multi-directional DSLM for confocal detection without striping artifacts

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Power-effective scanning with AODs for 3D optogenic applications

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks

Four-channel radio-frequency signal generator programmed by an open-source Arduino-based control system via single or quad Serial Peripheral Interface

Radio-frequency (RF) signal generators are standard laboratory equipment and a wide-range of open-source and commercial devices exists to address their many applications. Nonetheless, only few expensive and proprietary solutions can be re-configured within a wide frequency band and triggered on a micro-second timescale. Such specifications are required for applications that leverage variable radio-frequencies to generate programmed mixed signals, to control processes or states and to precisely steer laser beams using acousto-optical devices, tasks often needed in industrial manufacturing, atomic and molecular physics or microscopy.Here we present an open-source low-cost Arduino-based control system that can store up to millions of commands received from a computer and then perform reliable high-speed programming of an arbitrary device under its control (DUC) via a single or quad-wire Serial Peripheral Interface. The software architecture operates as a real-time state machine, making it easily extensible and adaptable to any DUC. Each configuration change can be triggered either externally or internally, reaching 1 MHz rates when using a Teensy 4.1 Arduino-compatible board. Leveraging this flexible system, we developed a programmable four-channel RF signal generator, based on an Analog Devices 9959 Evaluation board, and we demonstrated its capability and validated its performance.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Remote Observations in the Near Infrared

. We present our experiences in remote observations in Near Infrared bands operating a bidimensional instrument ARNICA. ARNICA, an infrared CCD detector operating at a telescope (TIRGO, Gornergrat, Switzerland) was controlled by an observer from Firenze, Italy. Despite the rather slow Internet link available, we were able to perform the observations in quite an acceptable way. The user interface process (a widget based X11 client) was executed locally on a Sun workstation. All processes responsible for hardware support (initialization of devices and their dynamic control and data acquisition itself) were executed remotely under DESQview/X on a PC dedicated to the control of ARNICA. The traffic was reduced to a minimum due to the truly distributed software used. In normal conditions this is just an exchange of short primitives which describe the task to be performed and of informative messages. There is also the possibility of a continuous display of the obtained images, with flexible c..

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acoustooptic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License