Aberrations of the point spread function of a multimode fiber

We investigate the point spread function of a multimode fiber. The distortion of the focal spot created on the fiber output facet is studied for a variety of the parameters. We develop a theoretical model of wavefront shaping through a multimode fiber and use it to confirm our experimental results and analyze the nature of the focal distortions. We show that aberration-free imaging with a large field of view can be achieved by using an appropriate number of segments on the spatial light modulator during the wavefront-shaping procedure. The results describe aberration limits for imaging with multimode fibers as in, e.g., microendoscopy.Comment: 10 pages, 6 figure

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo mouse brain tissue.Comment: 15 pages, 7 figure

Spectral Cross-Cumulants for Multicolor Super-resolved SOFI Imaging

Super-resolution optical fluctuation imaging (SOFI) provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species.Comment: main: 21 pages & 4 figures, supplementary 20 pages & 16 figure

High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

Descloux A, Mueller M, Navikas V, et al. High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism. Nanophotonics. 2020;9(1):143-148.Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with highspeed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells

Benchmarking of single imaging datasets

A method of estimating a value (311-313) indicative of a signal component of an input imaging dataset (190) includes determining a normalized Fourier-domain representation of the input imaging dataset (190); and for each one of multiple low-pass filters (320) having varying cut-off frequencies (301, 301-1, 301-2): determining the respective low-pass filtered normalized Fourier-domain representation of the input imaging dataset (190) based on the normalized Fourier-domain representation of the input imaging dataset and performing a respective comparison of the respective low-pass filtered normalized Fourier-domain representation of the input imaging dataset with the input imaging dataset; and based on results (302, 303) of the comparisons associated with the low-pass filters: estimating the value (311-313) indicative of the signal component of the input imaging dataset (190)

Development of novel experimental and computational methods for three-dimensional coherent and super-resolution microscopy

Optical microscopy is one widely used tool to study cell functions and the interaction of molecules at a sub-cellular level. Optical microscopy techniques can be broadly divided into two categories: partially coherent and incoherent. Coherent microscopy techniques are usually label-free and provide diffraction limited structural information about the sample refractive index distribution though the measurement of phase delay induced by the sample. Since they can be very conservative in the illumination power directed toward the sample, they exhibit very low photo-toxicity and are suitable for both high speed and time lapse imaging. Fluorescence microscopy is an incoherent microscopy method which uses biochemistry techniques to label cellular structures with fluorescent molecules which emit light when excited by a laser. Fluorescence microscopy provides diffraction limited high specificity imaging but is limited in time due to photo-bleaching and photo-toxicity. Super-resolution microscopy is a sub-category of fluorescence microscopy which manipulates and exploits some properties of the fluorescent molecules to achieve high specificity sub-diffraction imaging. Super-resolution comes however at the price of an increased total acquisition time, which limits the applications of super-resolution microscopy to relatively slow cellular processes. The ideal microscope however does not exist; due to the limited spatio-temporal bandwidth of far-field microscopy, there will always be unavoidable trade-offs. There is therefore a need to find new ways to ensure that the methods are reaching their optimal performances and a need to study how different methods can complement each others. I start by presenting a new method for three-dimensional quantitative phase retrieval. I derive a model for the image formation of three-dimensional bright field images and extrapolate a novel expression allowing to retrieve the phase distribution from a bright field image stack. Using a unique image-splitting multi-plane prism that allows to acquire 8 distinct focal planes in a single exposure, I demonstrate three-dimensional quantitative phase imaging at 200 Hz . Finally, I show the association of three-dimensional super-resolution SOFI with phase imaging. To improve the imaging speed and lower the illumination intensity, I combine the same prism platform with a high-speed structured illumination. Since the structured illumination presented is using a digital micro-mirror device, about 90 \% of the laser light is diffracted outside of the optical path. To improve the illumination efficiency but keep its speed and flexibility, I present a new approach for achromatic high power high speed SIM, based on a Michelson interferometer. With the access to high illumination power density, I also show the first experimental combination of SIM with SOFI using a self-blinking dye. Motivated by the absence of tools to objectively judge the performance of the microscopy methods I develop, I present a novel algorithm for image resolution estimation. The method estimate the resolution by correlating the image with several filtered version of itself without any external parameters. Finally, in the context of the AD-gut consortium, I show a practical application of deep neural networks used to assists the segmentation and mapping of the microscopy image of enzymatically labeled DNA molecules

Parameter-free image resolution estimation based on decorrelation analysis

Super-resolution microscopy opened diverse new avenues of research by overcoming the resolution limit imposed by diffraction. Exploitation of the fluorescent emission of individual fluorophores made it possible to reveal structures beyond the diffraction limit. To accurately determine the resolution achieved during imaging is challenging with existing metrics. Here, we propose a method for assessing the resolution of individual super-resolved images based on image partial phase autocorrelation. The algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in super-resolution microscopy

Experimental Combination of Super-Resolution Optical Fluctuation Imaging with Structured Illumination Microscopy for Large Fields-of-View

All fluorescence super-resolution microscopy techniques present trade-offs between, for example, resolution, acquisition speed, and live-cell compatibility. Structured illumination microscopy (SIM) improves the resolution through successive imaging of the sample under patterned illumination. SIM can be fast and typically uses low light levels well suited for live cell imaging. However, in its linear form, the resolution gain of SIM is limited by the pattern frequency to a 2-fold improvement over the diffraction limit. Super-resolution optical fluctuation imaging (SOFI) is another low-light level method that achieves higher resolution through the computation of spatiotemporal cross-cumulants of a time series of stochastically blinking fluorescent emitters. The resolution is theoretically enhanced by a factor n, where n is the cumulant order. In practice, it is restricted to smaller orders due to limited signal-to-noise and the need for many frames for good statistics. Here, we demonstrate the experimental combination of SOFT with SIM, where we use SOFI as a source of nonlinearity to further enhance the SIM resolution. We present two implementations of SIM combined with self-blinking dyes for SOFT. We first introduce a new Michelson SIM setup for achromatic high-efficiency (40%) illumination and fast structured pattern projection. We use the setup to acquire single- and two-color SIM data of blinking emitters with up to 2.4-fold image resolution increase and discuss the SOFI-SIM reconstruction challenges. We applied the same concept to realize SOFI-SIM on a flat-fielded, high-throughput instant SIM (iSIM) setup, achieving similar resolution enhancement and demonstrating the versatility of our approach. We established an experimental proof-of-principle of a wide-field combination of SOFT with SIM and iSIM for large fields-of-view, improving SIM resolution without increased complexity of the setup

Adaptive optics enables multimode 3D super-resolution microscopy via remote focusing

A variety of modern super-resolution microscopy methods provide researchers with previously inconceivable biological sample imaging opportunities at a molecular resolution. All of these techniques excel at imaging samples that are close to the coverslip, however imaging at large depths remains a challenge due to aberrations caused by the sample, diminishing the resolution of the microscope. Originating in astro-imaging, the adaptive optics (AO) approach for wavefront shaping using a deformable mirror is gaining momentum in modern microscopy as a convenient approach for wavefront control. AO has the ability not only to correct aberrations but also enables engineering of the PSF shape, allowing localization of the emitter axial position over several microns. In this study, we demonstrate remote focusing as another AO benefit for super-resolution microscopy. We show the ability to record volumetric data (45 × 45 × 10 μm), while keeping the sample axially stabilized using a standard widefield setup with an adaptive optics addon. We processed the data with single-molecule localization routines and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution. BN/Kristin Grussmayer La

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 μm axial and 0.4 μm lateral resolution maintained over a depth of 40 μm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.15 pages, 7 figuresstatus: publishe