We report a fast, easy-to-use adaptive optics (AO) method and its implementation in both Light-Sheet and Two-Photon fluorescence microscopy setups. We demonstrate fast (typically 1 second), and accurate aberrations correction at large depths (hundreds of microns) mostly in brain tissue of animal models such as Drosophila, Zebra Fish and mouse. We quantifiy the gain brought by AO in terms of signal (up to x5), contrast, achievable imaging depth and segmentation capability of neuronal structures for both imaging modalities. The approach paves the way to future automated AO-based 3D microscopy systems.
SignificanceAdaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method.AimProvide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples.ApproachDevelopment of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget.ResultsFast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO.ConclusionWe developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.
Optical microscopy allows to perform structural and functional imaging within large volume of tissues with subcellular resolution. Non-linear microscopy allows the interrogation of neuronal activity in mammalian brains but remains limited because of scattering and optical aberrations. To overcome these issues, Adaptive Optics (AO) strategies have been implemented to retrieve the microscope imaging quality while addressing important imaging depths.
A first AO strategy implemented in non-linear microscopy relies on a sensorless configuration, but is a time-consuming iterative process hardly compatible with photobleaching issues. A second approach is based on direct wavefront sensing using Shack-Hartmann wavefront sensors and has proved its efficiency on in vivo experiments. However, this method fails at large depths because of the strong scattering of the emitted fluorescence. A method for direct wavefront sensing more resilient to scattering of the fluorescence emission would therefore facilitate the use of AO in optical microscopy.
This work proposes an alternative method of direct wavefront measurement, which relies on the cross-correlation of images of an extended source obtained through a microlens array. This extended-source Shack-Hartmann wavefront sensor (ESSH) requires to be coupled to an optical sectioning method. Its efficiency has been proven when coupled to Light Sheet Fluorescence Microscopy in the adult drosophila brain in weekly scattering conditions. Here, we show that it allows quantitative aberration measurements through highly scattering fixed brain slices, up to four times the scattering length of the tissue. We demonstrate that it is more resilient to scattering compared to the current centroid-based approach. Taking advantage of its geometry, this new wavefront sensor also provides scattering coefficient measurements of biological tissues. Finally, we present its implementation on a two-photon microscope within a closed–loop configuration for in depth neuroimaging in mouse brain and compare its performances in scattering media to the classical centroid approach.
This conference presentation, “Axially-swept adaptive optics light sheet fluorescence microscopy for high resolution neuroimaging in the drosophila brain” was recorded for the Biomedical Spectroscopy, Microscopy, and Imaging II conference at SPIE Photonics Europe 2022.
We present a new implementation of adaptive optics for light-sheet microscopy, with a direct extended-scene wavefront sensing measurement for fast aberration correction. We report AO-enhanced images of GCaMP in freshly dissected drosophila brains.
We report a compact adaptive optics (AO) module with optimized optical design and photon budget, compatible with multiple wavelengths, and adaptable to most of existing Light-Sheet setups, enabling a 2 to 3-fold signal improvement on neuronal structures of the live, non-clarified drosophila brain (neurons, projections), at depths ranging from 50 to 100µm. We report similar signal improvement brought by AO on functional signals from neurons of the drosophila circadian clock network. The proposed setup paves the way to fully automatized AO Light-Sheet systems usable in routine by biologists.
Time-gated fluorescence imaging of near-infrared emitting ZnCuInSe/ZnS quantum dots (QDs) with fluorescence lifetimes in the range of 150−300 ns enables the efficient rejection of fast autofluorescence photons and the selection of QD fluorescence photons. This leads to complete elimination of autofluorescence background and a significant increase of imaging sensitivity. We demonstrate efficient detection and imaging of individual QD-labeled lymphoma cells circulating at mm/s velocities in blood vessels. In a second application, these QDs were used as a sensing platform to detect enzymatic activity using a ratiometric time-gated sensing scheme.
The in vivo detection of rare circulating cells using non invasive fluorescence imaging would provide a key tool to study migration of eg. tumoral or immunological cells. Fluorescence detection is however currently limited by a lack of contrast between the small emission of isolated, fast circulating cells and the strong autofluorescence background of the surrounding tissues. We present the development of near infrared emitting quantum dots (NIR-QDs) with long fluorescence lifetime for sensitive time-gated in vivo imaging of circulating cells. These QDs are composed of low toxicity ZnCuInSe/ZnS materials and made biocompatible using a novel multidentate imidazole zwitterionic block copolymer, ensuring their long term intracellular stability. Cells of interest can thus be labeled ex vivo with QDs, injected intravenously and imaged in the near infrared range. Excitation using a pulsed laser coupled to time-gated detection enables the efficient rejection of short lifetime (≈ ns) autofluorescence background and detection of long lifetime (≈ 150 ns) fluorescence from QD-labeled cells. We demonstrate efficient in vivo imaging of single fast-flowing cells, which opens opportunities for future biological studies.
[1] M. Tasso et al, “Sulfobetaine-Vinylimidazole block copolymers: a robust quantum dot surface chemistry expanding bioimaging’s horizons”, ACS Nano, 9(11), 2015
[2] S. Bouccara et al, “Time-gated cell imaging using long lifetime near-infrared-emitting quantum dots for autofluorescence rejection”, J Biomed Optc, 19(5), 2014
We present an implementation of a sensorless adaptive optics loop in a widefield fluorescence microscope. This setup is designed to compensate for aberrations induced by the sample on both excitation and emission pathways. It allows fast optical sectioning inside a living Drosophila brain. We present a detailed characterization of the system performances. We prove that the gain brought to optical sectioning by realizing structured illumination microscopy with adaptive optics down to 50 μm deep inside living Drosophila brain.
KEYWORDS: Luminescence, Near infrared, Quantum dots, Tissues, In vivo imaging, Signal detection, Pulsed laser operation, Fluorescence spectroscopy, Tissue optics, Microscopy
In vivo cell tracking is a promising tool to improve our understanding of certain biological processes (circulating tumor cell migration, immune cell activity). Several cell tracking techniques have been developed like MRI or PET but remain ill adapted to detect rare and individual cells because of their low spatial resolution and limited sensitivity. Fluorescence detection is a promising alternative. Its sensitivity is however limited by the high tissue autofluorescence and poor visible light penetration depth. To overcome these limitations, we have developed a novel cell imaging modality, based on nearinfrared quantum dots (QDs) allowing long term cell labeling and a sensitive detection based on time-gated wide field fluorescence microscopy. We present the synthesis and characterization of Zn-Cu-In-Se / ZnS (core/shell) QDs composed of low toxicity materials. These QDs exhibit a bright emission centered around 800 nm, where absorption and scattering of tissues are minimal. These nanocrystals are coated with a new surface chemistry, which yields small, stable, bright and individual probes in the cell cytoplasm for several days after the labeling. These QDs also present a fluorescence lifetime much longer (150-200 ns) than tissue autofluorescence (5-10 ns). By combining a pulsed excitation source to a time-gated fluorescence imaging system, we show that we can efficiently discriminate the QD signal from autofluorescence and thus increase the detection sensitivity of labeled cells into tissues.
Fluorescence imaging is a promising technique for the detection of individual cell migration. Its sensitivity is, however, limited by a high tissue autofluorescence and a poor visible light penetration depth. In order to solve this problem, the fluorescence signal peak wavelength should lie in an absorption and diffusion free region and should be distinguishable, either spectrally or temporally, from the autofluorescence background. We present, here, the synthesis and characterization of low toxicity Zn–Cu–In–Se/ZnS core/shell quantum dots. Their fluorescence emission wavelength peaks around 800 nm, where the absorption and scattering of tissues are minimal. They are coated with a new ligand, which yields small, stable, and bright individual probes in the live cell cytoplasm, even 48 h after the labeling. Furthermore, these near-infrared-emitting quantum dots have a long fluorescence lifetime component (around 150 ns) compared to autofluorescence (<5 ns ). Taking the advantage of this property and coupling these probes to a time-gated detection, we demonstrate efficiently the discrimination between the signal and short lifetime fluorescence such as the autofluorescence. This technique is supported by a method we developed, to massively stain cells that preserves the quantum dot stability and brightness for 48 h.
Optical Coherence Tomography (OCT) is an efficient technique for in-depth optical biopsy of biological tissues, relying
on interferometric selection of ballistic photons. Full-Field Optical Coherence Tomography (FF-OCT) is an alternative
approach to Fourier-domain OCT (spectral or swept-source), allowing parallel acquisition of en-face optical sections.
Using medium numerical aperture objective, it is possible to reach an isotropic resolution of about 1x1x1 ìm. After
stitching a grid of acquired images, FF-OCT gives access to the architecture of the tissue, for both macroscopic and
microscopic structures, in a non-invasive process, which makes the technique particularly suitable for applications in
pathology. Here we report a multimodal approach to FF-OCT, combining two Full-Field techniques for collecting a
backscattered endogeneous OCT image and a fluorescence exogeneous image in parallel. Considering pathological
diagnosis of cancer, visualization of cell nuclei is of paramount importance. OCT images, even for the highest resolution,
usually fail to identify individual nuclei due to the nature of the optical contrast used. We have built a multimodal optical
microscope based on the combination of FF-OCT and Structured Illumination Microscopy (SIM). We used x30 immersion objectives, with a numerical aperture of 1.05, allowing for sub-micron transverse resolution. Fluorescent staining of nuclei was obtained using specific fluorescent dyes such as acridine orange. We present multimodal images of healthy and pathological skin tissue at various scales. This instrumental development paves the way for improvements of standard pathology procedures, as a faster, non sacrificial, operator independent digital optical method compared to frozen sections.
We describe the implementation and use of an adaptive optics loop in the imaging path of a commercial wide field microscope. We show that it is possible to maintain the optical performances of the original microscope when imaging through aberrant biological samples. The sources used for illuminating the adaptive optics loop are spectrally independent, in excitation and emission, from the sample, so they do not appear in the final image, and their use does not contribute to the sample bleaching. Results are compared with equivalent images obtained with an identical microscope devoid of adaptive optics system.
Apertureless scanning near-field optical microscopy (SNOM) offers new opportunities in fluorescence imaging by providing subwavelength resolution. This is achieved by scattering the near-field with a metallic tip. SNOM images have been recorded on fluorescent spheres and erbium-doped vitroceramic. We will also present approach curves that allow to better understand the near-field optical contrast origin. Our near-field microscope is now suitable for immersed samples imaging, in order to study biological samples.
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