Adaptive optics light-sheet microscopy based on direct wavefront sensing without any guide star.

We propose an adaptive optics light-sheet fluorescence microscope (AO-LSFM) for closed-loop aberrations' correction at the emission path, providing intrinsic instrumental simplicity and high accuracy when compared to previously reported schemes. The approach is based on direct wavefront sensing, i.e., not on time-consuming iterative algorithms, and does not require the use of any guide star, thus reducing instrumental complexity and/or sample preparation constraints. The design is based on a modified Shack-Hartmann wavefront sensor providing compatibility with extended sources such as images from optical sectioning microscopes. We report an AO-LSFM setup based on such sensors, including characterization of the sensor performance, and demonstrate for the first time to the best of our knowledge a significant contrast improvement on neuronal structures of the ex vivo adult drosophila brain in depth.

Dynamics of mitochondrial membranes under photo-oxidative stress with high spatiotemporal resolution.

In our study, we harnessed an original Enhanced Speed Structured Illumination Microscopy (Fast-SIM) imaging setup to explore the dynamics of mitochondrial and inner membrane ultrastructure under specific photo-oxidation stress induced by Chlorin-e6 and light irradiation. Notably, our Fast-SIM system allowed us to observe and quantify a distinct remodeling and shortening of the mitochondrial structure after 60-80 s of irradiation. These changes were accompanied by fusion events of adjacent inner membrane cristae and global swelling of the organelle. Preceding these alterations, a larger sequence was characterized by heightened dynamics within the mitochondrial network, featuring events such as mitochondrial fission, rapid formation of tubular prolongations, and fluctuations in cristae structure. Our findings provide compelling evidence that, among enhanced-resolution microscopy techniques, Fast-SIM emerges as the most suitable approach for non-invasive dynamic studies of mitochondrial structure in living cells. For the first time, this approach allows quantitative and qualitative characterization of successive steps in the photo-induced oxidation process with sufficient spatial and temporal resolution.

Enhanced neuroimaging with a calcium sensor in ex-vivo Drosophila melanogaster brains using closed-loop adaptive optics light-sheet fluorescence microscopy.

Significance: Adaptive 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. Aim: Provide 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. Approach: Development 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. Results: Fast 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. Conclusion: We 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.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate.

In Vivo Imaging of Single Tumor Cells in Fast-Flowing Bloodstream Using Near-Infrared Quantum Dots and Time-Gated Imaging.

Whereas in vivo fluorescence imaging of cells immobilized within tissues provides a valuable tool to a broad range of biological studies, it still lacks the sensitivity required to visualize isolated cells circulating fast in the bloodstream due, in particular, to the autofluorescence from endogenous fluorophores. Time-gated 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, thus significantly increasing sensitivity. We labeled model erythrocytes as well as lymphoma cells using these QDs coated with a stable zwitterionic polymer surface chemistry. After reinjection in the bloodstream, we were able to image and count individual QD-labeled cells circulating at mm·s-1 velocities in blood vessels.

Sensorless adaptive optics implementation in widefield optical sectioning microscopy inside in vivo Drosophila brain.

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.

Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools.

Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD-protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD-protein A-antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 µM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD-protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions.

Time-gated cell imaging using long lifetime near-infrared-emitting quantum dots for autofluorescence rejection.

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.

Bayesian estimation for optimized structured illumination microscopy.

Structured illumination microscopy is a recent imaging technique that aims at going beyond the classical optical resolution by reconstructing high-resolution (HR) images from low-resolution (LR) images acquired through modulation of the transfer function of the microscope. The classical implementation has a number of drawbacks, such as requiring a large number of images to be acquired and parameters to be manually set in an ad-hoc manner that have, until now, hampered its wide dissemination. Here, we present a new framework based on a Bayesian inverse problem formulation approach that enables the computation of one HR image from a reduced number of LR images and has no specific constraints on the modulation. Moreover, it permits to automatically estimate the optimal reconstruction hyperparameters and to compute an uncertainty bound on the estimated values. We demonstrate through numerical evaluations on simulated data and examples on real microscopy data that our approach represents a decisive advance for a wider use of HR microscopy through structured illumination.

Spectroscopy of single CdSe nanoplatelets.

We collect and resolve spectrally and temporally the photoluminescence of single CdSe nanoplatelets. The emission intensity of single nanoplatelets at room temperature shows ON and OFF periods with a usual blinking statistics, while at 20 K, their emission intensity can be extremely stable in time. At room temperature, the emission spectra of single nanoplatelets are similar to ensemble measurements with a full width at half-maximum of 40 meV. At 20 K, we obtain a resolution-limited spectral line width (<0.4 meV). The fluorescence lifetime of single nanoplatelets decreases when the temperature decreases to reach 200 ps at 20 K. This lifetime shortening is concomitant with an increase of the nanoplatelets' emission intensity.

Adaptive optics for fluorescence wide-field microscopy using spectrally independent guide star and markers.

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.

Axial coding in full-field microscopy using three-dimensional structured illumination implemented with no moving parts.

We report a simple optical setup to produce both axial and lateral structured illumination through a single objective lens. With a minimum of six full-field images obtained without moving either the sample or the microscope objective, 100 nm diameter fluorescent beads can be localized axially with an accuracy of 50 nm in a 1.76-microm-thick layer. We show that this axial localization improvement can easily be combined with classical lateral structured illumination, so that lateral resolution enhancement by a factor of 2 is maintained.

Absorption of low-loss optical materials measured at 1064 nm by a position-modulated collinear photothermal detection technique.

A collinear photothermal detection bench is described that makes use of a position-modulated heating source instead of the classic power-modulated source. This new modulation scheme increases by almost a factor 2 the sensitivity of a standard mirage bench. This bench is then used to measure the absorption coefficient of OH-free synthetic fused silica at 1064 nm in the parts per 10(6) range, which, combined with spectrophotometric measurements, confirms that the dominant absorption source is the OH content.

Confocal thermal-lens microscope.

The use of a confocal detection scheme in a dual-beam thermal-lens microscope is presented. The scheme allows the measurement of absorption factors down to 1.2 x 10(-7) in a 0.35-microm3 volume by use of a heating laser power of 100 mW incident upon the sample. Results are presented that prove that a 450-nm axial resolution is possible when a 1.2 water immersion objective lens with a N.A. of 1.2 is used.

Full-field birefringence imaging by thermal-light polarization-sensitive optical coherence tomography. I. Theory.

A method for measuring birefringence by use of thermal-light polarization-sensitive optical coherence tomography is presented. The use of thermal light brings to polarization-sensitive optical coherence tomography a resolution in the micrometer range in three dimensions. The instrument is based on a Linnik interference microscope and makes use of achromatic quarter-wave plates. A mathematical representation of the instrument is presented here, and the detection scheme is described, together with a discussion of the validity domain of the equations used to evaluate the birefringence in the presence of white-light illumination.

Full-field birefringence imaging by thermal-light polarization-sensitive optical coherence tomography. II. Instrument and results.

We describe an instrument for measuring the magnitude of birefringence of tomographic images and the principal directions of axes that use thermal-light polarization-sensitive optical coherence tomography. The instrument permits full-field measurements with an axial resolution of 1.5 microm and a transverse resolution limited by diffraction. We obtained a sensitivity of 84 dB, limited by shot noise, when we integrated the signal for 1 s. We verified the validity of the measurement by measuring the birefringence of a variable phase shifter. We present typical results obtained with optical samples.