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The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an extended depth of field (EDF) approach in a random illumination microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets.

Controlling the propagation and interaction of light in complex media has sparked major interest in the past few years. Unfortunately, spatial light modulation devices suffer from limited speed, which precludes real-time applications such as imaging in live tissue. To address this critical problem, we introduce a phase-control technique to characterize complex media based on the use of fast one-dimensional (1D) spatial light modulators and a 1D-to-2D transformation performed by the same medium being analysed. We implement the concept using a microelectromechanical grating light valve with 1,088 degrees of freedom, modulated at 350 kHz, enabling unprecedented high-speed wavefront measurements. We continuously measure the transmission matrix, calculate the optimal wavefront and project a focus through various dynamic scattering samples in real time, all within 2.4 ms per cycle. These results improve the previously achieved wavefront shaping modulation speed by more than an order of magnitude and open new opportunities for optical processing using 1D-to-2D transformations.

Coherent Raman microscopy is the method of choice for the label-free, real-time characterization of the chemical composition in biomedical samples. The common implementation relies on scanning two tightly focused laser beams across the sample, which frequently leads to sample damage and proves slow over large fields of view. The few existing wide-field techniques, for their part, feature a reduced lateral resolution and do not provide axial sectioning. To resolve these practical limitations, we developed a robust wide-field nonlinear microscope that combines random illumination microscopy (RIM) with coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG) contrasts. Based on a comprehensive theoretical study, CARS-RIM provides super-resolved reconstructions and optical sectioning of the sample from the second-order statistics of multiple images obtained under different speckled illuminations. We experimentally show that multimodal CARS-RIM and SFG-RIM achieve wide-field nonlinear imaging with a 3 µm axial sectioning capability and a 300 nm transverse resolution, effectively reducing the peak intensity at the sample compared with conventional point-scanning CARS. We exemplify the label-free, highly contrasted chemical imaging potential of CARS-RIM and SFG-RIM wide-field microscopy in two dimensions, as well as three dimensions, for a variety of samples such as beads, unstained human breast tissue and a mixture of chemical compounds.Combining random illumination microscopy with coherent anti-Stokes Raman scattering and sum-frequency generation contrasts, a robust wide-field nonlinear microscope with a 3 µm axial sectioning capability and a 300 nm transverse resolution is demonstrated.

Confocal and multiphoton microscopy are effective techniques to obtain high-contrast images of 2-D sections within bulk tissue. However, scattering limits their application to depths only up to ~1 millimeter. Multimode fibers make excellent ultrathin endoscopes that can penetrate deep inside the tissue with minimal damage. Here, we present Multiview Scattering Scanning Imaging Confocal (MUSSIC) Microscopy that enables high signal-to-noise ratio (SNR) imaging through a multimode fiber, hence combining the optical sectioning and resolution gain of confocal microscopy with the minimally invasive penetration capability of multimode fibers. The key advance presented here is the high SNR image reconstruction enabled by employing multiple coplanar virtual pinholes to capture multiple perspectives of the object, re-shifting them appropriately and combining them to obtain a high-contrast and high-resolution confocal image. We present the theory for the gain in contrast and resolution in MUSSIC microscopy and validate the concept through experimental results.

Diffraction-limited imaging in epi-fluorescence microscopy remains a challenge when sample aberrations are present or when the region of interest rests deep within an inhomogeneous medium. Adaptive optics is an attractive solution albeit with limited field of view and requiring relatively complicated systems. Alternatively, reconstruction algorithms have been developed over the years to correct for aberrations. Unfortunately, purely postprocessing techniques tend to be ill-posed and provide only incremental improvements in image quality. Here, we report a computational optical approach using unknown speckle illumination and matched reconstruction algorithm to correct for aberrations and reach or surpass diffraction limited resolution. The data acquisition is performed by shifting an unknown speckle pattern with respect to the fluorescent object. The method recovers simultaneously a high-resolution image, the point spread function of the system that contains the aberrations, the speckle illumination pattern, and the shift positions.

Improving the resolution of fluorescence microscopy beyond the diffraction limit can be achievedby acquiring and processing multiple images of the sample under different illumination conditions.One of the simplest techniques, Random Illumination Microscopy (RIM), forms the super-resolvedimage from the variance of images obtained with random speckled illuminations. However, thevalidity of this process has not been fully theorized. In this work, we characterize mathematicallythe sample information contained in the variance of diffraction-limited speckled images as a functionof the statistical properties of the illuminations. We show that an unambiguous two-fold resolutiongain is obtained when the speckle correlation length coincides with the width of the observationpoint spread function. Last, we analyze the difference between the variance-based techniques usingrandom speckled illuminations (as in RIM) and those obtained using random fluorophore activation(as in Super-resolution Optical Fluctuation Imaging, SOFI).

Recently, it has been shown theoretically that fluorescence microscopy using random illuminations (RIM) yields a doubled lateral resolution and an improved optical sectioning. Moreover, an algorithm called algoRIM, based on variance matching, has been successfully validated on numerous biological applications. Here, we propose a proof of uniqueness of the RIM variance equation, which corresponds to a first theoretical validation of algoRIM.

Controlling the propagation and interaction of light in complex media has sparked major interest in the last few years. Unfortunately, spatial light modulation devices suffer from limited speed that precludes real-time applications such as imaging in live tissue. To address this critical problem we introduce a phase-control technique to characterize complex media based on the use of fast 1D spatial light modulators and a 1D-to-2D transformation performed by the same medium being analyzed. We implement the concept using a micro-electro-mechanical grating light valve (GLV) with 1088 degrees of freedom modulated at 350 KHz, enabling unprecedented high-speed wavefront measurements. We continuously measure the transmission matrix, calculate the optimal wavefront and project a focus through various dynamic scattering samples in real-time, all within 2.4 ms per cycle. These results improve prior wavefront shaping modulation speed by more than an order of magnitude and open new opportunities for optical processing using 1D-to-2D transformations.

Speckle based imaging consists of forming a super-resolved reconstruction of an unknown sample from low-resolution images obtained under random inhomogeneous illuminations (speckles). In a blind context where the illuminations are unknown, we study the intrinsic capacity of speckle-based imagers to recover spatial frequencies outside the frequency support of the data, with minimal assumptions about the sample. We demonstrate that, under physically realistic conditions, the covariance of the data has a super-resolution power corresponding to the squared magnitude of the imager point spread function. This theoretical result is important for many practical imaging systems such as acoustic and electromagnetic tomographs, fluorescence and photoacoustic microscopes, or synthetic aperture radar imaging. A numerical validation is presented in the case of fluorescence microscopy.

The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an Extended Depth of Field (EDF) approach in a Random Illumination Microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets.