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As a remarkable answer to the growing interest in rapid 3D visualization  of biological samples in vivo, Light-sheet Fluorescence Microscopy (LSFM) has become a valuable tool for biologists. Indeed, it enables optical sectioning of a sample with low photo-toxicity and low photo-bleaching at a very fast imaging rate. Many implementations of LSFM now exist, with a great number of commercial and home-made solutions. Among all those instruments, Lattice Light-Sheet Microscopy (LLSM) stands out as one of the most efficient techniques for fast 3D imaging at sub-cellular scale. However, sample-induced aberrations are still limiting for in-depth observations inside thick tissues.

Active Image Optimization (AIO) enables deeper and better resolved LLSM

To get rid of these aberrations, Adaptive Optics (AO) was integrated in some LLSM setups, providing signal and resolution enhancement, but often at the cost of much more complex systems. In order to propose a simpler and cheaper solution, a team of researchers from Université de Bordeaux (CNRS, France) and Imagine Optic recently developed an original approach, so-called Active Image Optimization (AIO) (full publication here : https://doi.org/10.1364/BOE.471757).

The AIO method is based on two steps : first, an original light-sheet auto-focus process using a sequence of sample images ensures accurate coplanarity between the illumination & imaging planes, then a sensorless, image-based iterative AO optimization  is performed, providing aberration correction at the emission path. Based on this method, the authors determined an optimal merit factor for their samples of interest, i.e. fixed organotypic mouse brain slices. As a proof of the efficiency of AIO, researchers were able to retrieve normal average spine head sizes down to 40 µm, as compared to enlarged structures imaged without AIO.

The developed setup corresponds to both hardware and software add-ons to a standard LLSM system, based on AO kit Bio, a set of adaptive optics components (HASO 4 First Shack-Hartmann WaveFront Sensor, MirAO 52-e deformable mirror and Wavekit Bio SDK) from Imagine Optic. This kit can be used in a various range of set-ups, such as in, for example, a conventional Light-Sheet microscope.

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When targeting high-resolution imaging of biological samples at large depths, non-linear microscopy, and in particular 2- or even 3-photon fluorescence microscopy, is usually a technique of choice. Due to near-infrared illumination and intrinsic optical sectioning capability, multiphoton microscopy provides deep 3D imaging with virtually no background and a great versatility enabled by the use of 2D scanning in the illumination path. This method is now widely employed for neuroimaging, particularly in scattering media such as rodent brain. However, as for all high-resolution optical microscopy techniques, its performance is severely reduced as a consequence of optical aberrations induced by the sample, especially in depth, which strongly degrades the illumination point spread function (PSF) resulting in a significant loss of signal and contrast.

Recently, we demonstrated a new, fast and simple AO approach, as well as its integration in a light-sheet microscope (more details here). This new AO method is based on direct wavefront sensing without the need for a guide star, enabling both fast correction (typ. 1 to 5 s) and reduction of the constraints of use. Aiming to provide users with an easy operation of AO in multiple microscopy modalities, Imagine Optic, together with a team of researchers from Ecole Supérieure de Physique et de Chimie Industrielle (ESPCI, France) and Ecole Normale Supérieure (ENS, France), adapted AO to the excitation path of a custom-built two photon microscope. This wavefront sensing approach was demonstrated to be particularly efficient in scattering conditions (publication here). 

Our prototype microscope is using one of Mirao line of large stroke, high stability deformable mirrors and allowed us to acquire first images   of ex vivo samples mainly consisting in fixed fluorescent mouse brain slices, at depths reaching 200µm (see upper figure, representing a maximum projection over 10µm brain slice). Only 4 iterations of the closed-loop optimization, corresponding to 2-3 seconds, were necessary to achieve this aberration correction. AO correction enabled an average three times increase of the signal, providing sharper morphological details such as axons or dendrites. These latest results have been recently presented at the SFO congress in Nice (France): Imperato S. et al., Extended scene adaptive optics for 2 photon Neuroimaging in the mouse brain, 05 Jul. 2022. They offer a great promise, specifically regarding the next steps of in vivo recording of functional signals, as well as product development.

These results have been achieved in the frame of the INOVAO project (funding Agence Nationale de la Recherche, ANR-18-CE19-0002).

#AdaptiveOptics #Microscopy

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Light-sheet fluorescence microscopy proved many advantages when imaging biological samples, providing high speed, low phototoxicity, large field of view, 3D capability together with optical sectioning. However, and in particular when imaging deep, sample-induced optical aberrations usually limit the image quality. Adaptive optics (AO) can compensate for such aberrations, but currently at the expense of either slow speed or complicated setups, which are not yet ready to be used routinely.

With the aim to provide a systematic benefit of using adaptive optics in light-sheet microscopy, Imagine Optic, together with a team of researchers from Ecole Supérieure de Physique et de Chimie Industrielle (ESPCI, France), developed a prototype of adaptive optics light-sheet fluorescence microscope. To compensate for sample-induced aberrations, this microscope contains a deformable mirror on the emission pathway and it can benefit from both sensorless and direct wavefront sensing-based adaptive optics approaches. The latter technology is based on our newly developed wavefront sensing approach (full description here) which enables fast adaptive optics correction – typically within 1 to 3 seconds – without the need for a guide star in the sample.

This new aberration detection and correction approach already demonstrated a significant improvement of the image quality in neuroscience samples (see an example here and in the previously mentioned publication). With this microscope setup we are now able to demonstrate and quantify the gain brought by adaptive optics in light-sheet microscopy for various biological samples. In particular it provides significant signal increase when imaging small structures close to the diffraction limit, especially when imaging deep, and/or when small signals need to be observed with a decent signal-to-noise ratio. Everybody who is interested in discussing AO for light-sheet and/or testing their samples are welcome to contact us.

These results have been achieved in the frame of the INOVAO project (funding Agence Nationale de la Recherche, ANR-18-CE19-0002).

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Adaptive Optics approaches in fluorescence microscopy

Although many Adaptive Optics (AO) approaches have been proposed in fluorescence microscopy, the presence of scattering deeper in biological samples, typically dictates the use of sensorless AO methods. In situations where scattering severely limits the use of a guide star, either based on fluorescent beads or involving a de-scan of fluorescence signal to enable the correction of optical aberrations, a classical Shack-Hartmann wavefront sensor cannot be used. However, sensorless AO – even if instrumentally simpler – provides a correction of aberrations at the cost of time-consuming iterative process of typically tens of seconds. Moreover, the quality of the correction is also driven by algorithmic parameters such as the choice of an adequate image quality metric. 

Direct wavefront sensing for more accurate AO in microscopy

As a new step towards a faster and more accurate AO in microscopy even in scattering conditions, a team of researchers from Ecole Supérieure de Physique et de Chimie Industrielle (ESPCI, France), Ecole Normale Supérieure (ENS, France) and Imagine Optic recently proposed the use of an extended-source Shack-Hartmann wavefront sensor as a direct wavefront sensing device, which is more resilient to scattering than existing methods (full publication here). Even in low-signal to background conditions, a successful AO correction was demonstrated deep in the brain tissue in less than a second. Interestingly, researchers demonstrated that this device can also be used to quantitatively characterize the scattering properties of the sample. 

As for all AO setups, the proposed method benefits from the almost perfect linearity of the wavefront corrector, in this case the Mirao 52e electromagnetic deformable mirror [FH1] , as well as from its high dynamic range and intrinsic achromaticity, e.g. considering its use in 2-photon microscopy setups. Keep posted to know more about our upcoming compact, high-resolution deformable mirror (µ-DM), that was recently presented here. 

These results have been achieved in the frame of the INOVAO project (funding Agence Nationale de la Recherche, ANR-18-CE19-0002). #adaptive optics microscopy direct wavefront sensing

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We recently communicated the latest advances and results about this method and other implementations of our adaptive optics technology in microscopy at Focus on Microscopy, March 28-31 2021: the 5 talks we contributed to detailed how adaptive optics can boost imaging performance in Light-Sheet and Single-Molecule Localization Microscopy. 

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