Video-rate volumetric neuronal imaging using 3D targeted illumination

Fast volumetric microscopy is required to monitor large-scale neural ensembles with high spatio-temporal resolution. Widefield fluorescence microscopy can image large 2D fields of view at high resolution and speed while remaining simple and costeffective. A focal sweep add-on can further extend the capacity of widefield microscopy by enabling extended-depth-of-field (EDOF) imaging, but suffers from an inability to reject out-of-focus fluorescence background. Here, by using a digital micromirror device to target only in-focus sample features, we perform EDOF imaging with greatly enhanced contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. We demonstrate the advantages of our technique for in vivo calcium imaging in the mouse brain.This work was funded by the National Institutes of Health (R21EY026310) and the National Science Foundation (CBET-1508988). The authors wish to thank E. McCarthy and Prof. M.J. Baum for providing mouse brain slices used in this manuscript, and A. I. Mohammed for providing in vivo mouse brain samples in the early stages of this work. (R21EY026310 - National Institutes of Health; CBET-1508988 - National Science Foundation)Published versio

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume

Superresolution fluorescence microscopy with structured illumination

The resolution of a conventional fluorescence microscope image is diffraction limited which achieves a spatial resolution of 200nm lateral and 500nm axial. Recently, many superresolution fluorescence microscopy techniques have been developed which allow the observation of many biological structures beyond the diffraction limit. Structured illumination microscopy (SIM) is one of them. The principle of SIM is based on using a harmonic light grid which down modulates the high spatial frequencies of the sample into the observable region of the microscope. The resolution enhancement is highly dependent on the reconstruction technique, which restores the high spatial frequencies of the sample to their original position. Common SIM reconstructions require the perfect knowledge of the illumination pattern. However, to perfectly control the harmonic illumination patterns on the sample plane is not easy in experimental implementations and this makes the experimental setup very technical. Reconstructing SIM images assuming the perfect knowledge of the illumination intensity patterns may, therefore, introduce artifacts on the estimated sample due to the misalignment of the grid that can occur during experimental acquisitions. To tackle this drawback of SIM, in this these, we have developed blind-SIM reconstruction strategies which are independent of the illumination patterns. Using the 3D blind-SIM reconstruction strategies we extended the harmonic SIM to speckle illumination microscopy which uses random unknown speckle patterns that need no control, unlike the harmonic grid patterns. For harmonic-SIM images, since incorporating some information about illumination patterns is valuable, we have developed a 3D positive filtered blind-SIM reconstruction which confines the iterative estimation of the illuminations in the vicinity of the Fourier peaks (using carefully designed Fourier filter masks) in the Fourier space. Using blind-SIM reconstruction techniques a lateral resolution of about 100nm and axial resolution of about 200nm is obtained in both speckle and harmonic SIM. In addition, to reduce the out-of-focus problem in widefield images, a simple computational technique which is based on reconstructing 2D data with 3D PSF is developed based on blind-SIM reconstruction. Moreover, to combine the functionalities of SIM and light sheet microscopy, as a proof of concept, we have developed a simple microscope setup which produces a structured light sheet illumination pattern.La microscopie de fluorescence optique est l’un des outils les plus puissants pour étudier les structures cellulaires et moléculaires au niveau subcellulaire. La résolution d’une image de microscope conventionnel à fluorescence est limitée par la diffraction, ce qui permet d’obtenir une résolution spatiale latérale de 200nm et axiale de 500nm. Récemment, de nombreuses techniques de microscopie de fluorescence de super-résolution ont été développées pour permettre d’observer de nombreuses structures biologiques au-delà de la limite de diffraction. La microscopie d’illumination structurée (SIM) est l’une de ces technologies. Le principe de la SIM est basé sur l’utilisation d’une grille de lumière harmonique qui permet de translater les hautes fréquences spatiales de l’échantillon vers la région d’observation du microscope. L’amélioration de la résolution de cette technologie de microscopie dépend fortement de la technique de reconstruction, qui rétablit les hautes fréquences spatiales de l’échantillon dans leur position d’origine. Les méthodes classiques de reconstruction SIM nécessitent une connaissance parfaite de l’illumination de l’échantillon. Cependant, l’implémentation d’un contrôle parfait de l’illumination harmonique sur le plan de l’échantillon n’est pas facile expérimentalement et il présente un grand défi. L’hypothèse de la connaissance parfaite de l’intensité de la lumière illuminant l’échantillon en SIM peut donc introduire des artefacts sur l’image reconstruite de l’échantillon, à cause des erreurs d’alignement de la grille qui peuvent se présenter lors de l’acquisition expérimentale. Afin de surmonter ce défi, nous avons développé dans cette thèse des stratégies de reconstruction «aveugle» qui sont indépendantes de d’illumination. À l’aide de ces stratégies de reconstruction dites «blind-SIM», nous avons étendu la SIM harmonique pour l’appliquer aux cas de «SIM-speckle» qui utilisent des illuminations aléatoires et inconnues qui contrairement à l’illumination harmonique, ne nécessitent pas de controle. Comme il est utile de récupérer des informations sur l’illumination en SIM harmonique, nous avons développé une reconstruction blind-SIM tridimensionnel et filtrée qui confine l’estimation itérative des illuminations au voisinage des pics dans l’espace de Fourier, en utilisant des masques de filtre de Fourier soigneusement conçus. En utilisant des techniques de reconstruction blind-SIM, une résolution latérale d’environ 100 nm et une résolution axiale d’environ 200 nm sont obtenues, à la fois en SIM harmonique et en SIM speckle. En outre, pour réduire le problème de focalisation dans les images de champ large, une technique de calcul simple qui repose sur la reconstruction bidimensionnel de données à partir de PSF tridimensionnel est développée. En outre, afin de combiner à la fois les fonctionnalités de la SIM et de la microscopie á nappe de lumière, en tant que preuve de concept, nous avons développé une configuration de microscope simple qui produit une nappe de lumière structuré

Superresolution fluorescence microscopy with structured illumination

Cotutela Universitat Politècnica de Catalunya i Aix-Marseille UniversitéThe resolution of a conventional fluorescence microscope image is diffraction limited which achieves a spatial resolution of 200nm lateral and 500nm axial. Recently, many superresolution fluorescence microscopy techniques have been developed which allow the observation of many biological structures beyond the diffraction limit. Structured illumination microscopy (SIM) is one of them. The principle of SIM is based on using a harmonic light grid which down modulates the high spatial frequencies of the sample into the observable region of the microscope. The resolution enhancement is highly dependent on the reconstruction technique, which restores the high spatial frequencies of the sample to their original position. Common SIM reconstructions require the perfect knowledge of the illumination pattern. However, to perfectly control the harmonic illumination patterns on the sample plane is not easy in experimental implementations and this makes the experimental setup very technical. Reconstructing SIM images assuming the perfect knowledge of the illumination intensity patterns may, therefore, introduce artifacts on the estimated sample due to the misalignment of the grid that can occur during experimental acquisitions. To tackle this drawback of SIM, in this these, we have developed blind-SIM reconstruction strategies which are independent of the illumination patterns. Using the 3D blind-SIM reconstruction strategies we extended the harmonic SIM to speckle illumination microscopy which uses random unknown speckle patterns that need no control, unlike the harmonic grid patterns. For harmonic-SIM images, since incorporating some information about illumination patterns is valuable, we have developed a 3D positive filtered blind-SIM reconstruction which confines the iterative estimation of the illuminations in the vicinity of the Fourier peaks (using carefully designed Fourier filter masks) in the Fourier space. Using blind-SIM reconstruction techniques a lateral resolution of about 100nm and axial resolution of about 200nm is obtained in both speckle and harmonic SIM. In addition, to reduce the out-of-focus problem in widefield images, a simple computational technique which is based on reconstructing 2D data with 3D PSF is developed based on blind-SIM reconstruction. Moreover, to combine the functionalities of SIM and light sheet microscopy, as a proof of concept, we have developed a simple microscope setup which produces a structured light sheet illumination pattern.La microscopie de fluorescence optique est l’un des outils les plus puissants pour étudier les structures cellulaires et moléculaires au niveau subcellulaire. La résolution d’une image de microscope conventionnel à fluorescence est limitée par la diffraction, ce qui permet d’obtenir une résolution spatiale latérale de 200nm et axiale de 500nm. Récemment, de nombreuses techniques de microscopie de fluorescence de super-résolution ont été développées pour permettre d’observer de nombreuses structures biologiques au-delà de la limite de diffraction. La microscopie d’illumination structurée (SIM) est l’une de ces technologies. Le principe de la SIM est basé sur l’utilisation d’une grille de lumière harmonique qui permet de translater les hautes fréquences spatiales de l’échantillon vers la région d’observation du microscope. L’amélioration de la résolution de cette technologie de microscopie dépend fortement de la technique de reconstruction, qui rétablit les hautes fréquences spatiales de l’échantillon dans leur position d’origine. Les méthodes classiques de reconstruction SIM nécessitent une connaissance parfaite de l’illumination de l’échantillon. Cependant, l’implémentation d’un contrôle parfait de l’illumination harmonique sur le plan de l’échantillon n’est pas facile expérimentalement et il présente un grand défi. L’hypothèse de la connaissance parfaite de l’intensité de la lumière illuminant l’échantillon en SIM peut donc introduire des artefacts sur l’image reconstruite de l’échantillon, à cause des erreurs d’alignement de la grille qui peuvent se présenter lors de l’acquisition expérimentale. Afin de surmonter ce défi, nous avons développé dans cette thèse des stratégies de reconstruction «aveugle» qui sont indépendantes de d’illumination. À l’aide de ces stratégies de reconstruction dites «blind-SIM», nous avons étendu la SIM harmonique pour l’appliquer aux cas de «SIM-speckle» qui utilisent des illuminations aléatoires et inconnues qui contrairement à l’illumination harmonique, ne nécessitent pas de controle. Comme il est utile de récupérer des informations sur l’illumination en SIM harmonique, nous avons développé une reconstruction blind-SIM tridimensionnel et filtrée qui confine l’estimation itérative des illuminations au voisinage des pics dans l’espace de Fourier, en utilisant des masques de filtre de Fourier soigneusement conçus. En utilisant des techniques de reconstruction blind-SIM, une résolution latérale d’environ 100 nm et une résolution axiale d’environ 200 nm sont obtenues, à la fois en SIM harmonique et en SIM speckle. En outre, pour réduire le problème de focalisation dans les images de champ large, une technique de calcul simple qui repose sur la reconstruction bidimensionnel de données à partir de PSF tridimensionnel est développée. En outre, afin de combiner à la fois les fonctionnalités de la SIM et de la microscopie á nappe de lumière, en tant que preuve de concept, nous avons développé une configuration de microscope simple qui produit une nappe de lumière structuréePostprint (published version

High-speed in vitro intensity diffraction tomography

We demonstrate a label-free, scan-free intensity diffraction tomography technique utilizing annular illumination (aIDT) to rapidly characterize large-volume three-dimensional (3-D) refractive index distributions in vitro. By optimally matching the illumination geometry to the microscope pupil, our technique reduces the data requirement by 60 times to achieve high-speed 10-Hz volume rates. Using eight intensity images, we recover volumes of ∼350 μm  ×  100 μm  ×  20  μm, with near diffraction-limited lateral resolution of   ∼  487  nm and axial resolution of   ∼  3.4  μm. The attained large volume rate and high-resolution enable 3-D quantitative phase imaging of complex living biological samples across multiple length scales. We demonstrate aIDT’s capabilities on unicellular diatom microalgae, epithelial buccal cell clusters with native bacteria, and live Caenorhabditis elegans specimens. Within these samples, we recover macroscale cellular structures, subcellular organelles, and dynamic micro-organism tissues with minimal motion artifacts. Quantifying such features has significant utility in oncology, immunology, and cellular pathophysiology, where these morphological features are evaluated for changes in the presence of disease, parasites, and new drug treatments. Finally, we simulate the aIDT system to highlight the accuracy and sensitivity of the proposed technique. aIDT shows promise as a powerful high-speed, label-free computational microscopy approach for applications where natural imaging is required to evaluate environmental effects on a sample in real time.https://arxiv.org/abs/1904.06004Accepted manuscrip

THREE-DIMENSIONAL (3D) IMAGE FORMATION AND RECONSTRUCTION FOR STRUCTURED ILLUMINATION MICROSCOPY USING A 3D TUNABLE PATTERN

Specific needs in live-cell microscopy necessitate moving fluorescence microscopy toward 3D imaging with enhanced spatial and temporal resolution. Exciting the sample by non-uniform illumination instead of uniform illumination is the main idea of developing techniques to address Abbes diffraction limit in the conventional widefield fluorescence microscopy. In this dissertation, we characterized a novel tunable structured illumination microscopy (SIM) system using a Fresnel biprism illuminated by multiple linear incoherent sources (slits), in which the lateral and axial modulation frequencies of the 3D structured illumination (SI) pattern can be tuned separately. This is a unique feature, which is not the case of the conventional SIM systems. First, in order to take advantage of the tunable-frequency 2D-SIM system (using a single slit), we present a computational approach to reconstruct optical-sectioned images with super-resolution enhancement (OS-SR) by combining data from two lateral modulation frequencies. Moreover, a computational approach to reduce residual fringes evident in the restored images from the Fresnel biprism-based incoherent tunable SIM system is proposed. Second, the 3D SI pattern and the forward imaging model for the tunable-frequency 3D-SIM system (using multiple slits) are verified experimentally and two reconstruction methods have been used to evaluate the achieved OS and SR capabilities. Third, we presented the design of the 3D SI system used in a tunable 3D-SIM setup and discussed its performance in terms of synthetic optical transfer function (OTF). By designing the slit element, we can engineer the frequency response of our 3D-SIM system to always operate at the highest OS and SR performance for a given imaging application. This is the first 3D-SIM setup that enables independent control of the achieved OS and SR capabilities. Finally, we proposed and implemented a new 3D iterative deconvolution approach based on a model that takes into account the axial scanning of the specimen during the data acquisition as in commercial microscopes. The method minimizes the mean squared error using the conjugate gradient descent optimization method. To our knowledge, such a restoration method has not been published to date