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Quantitative phase microscopies (QPMs) play a pivotal role in bio-imaging, offering unique insights that complement fluorescence imaging. They provide essential data on mass distribution and transport, inaccessible to fluorescence techniques. Additionally, QPMs are label-free, eliminating concerns of photobleaching and phototoxicity. However, navigating through the array of available QPM techniques can be complex, making it challenging to select the most suitable one for a particular application. This tutorial review presents a thorough comparison of the main QPM techniques, focusing on their accuracy in terms of measurement precision and trueness. We focus on 8 techniques, namely digital holographic microscopy (DHM), cross-grating wavefront microscopy (CGM), which is based on QLSI (quadriwave lateral shearing interferometry), diffraction phase microscopy (DPM), differential phase-contrast (DPC) microscopy, phase-shifting interferometry (PSI) imaging, Fourier phase microscopy (FPM), spatial light interference microscopy (SLIM), and transport-of-intensity equation (TIE) imaging. For this purpose, we used a home-made numerical toolbox based on discrete dipole approximation (IF-DDA). This toolbox is designed to compute the electromagnetic field at the sample plane of a microscope, irrespective of the object’s complexity or the illumination conditions. We upgraded this toolbox to enable it to model any type of QPM, and to take into account shot noise. In a nutshell, the results show that DHM and PSI are inherently free from artefacts and rather suffer from coherent noise; In CGM, DPC, DPM and TIE, there is a trade-off between precision and trueness, which can be balanced by varying one experimental parameter; FPM and SLIM suffer from inherent artefacts that cannot be discarded experimentally in most cases, making the techniques not quantitative especially for large objects covering a large part of the field of view, such as eukaryotic cells.

The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out-of-focus fluorescence and diffraction limited resolution. In this work, we show that a single slice of Structured Illumination Microscopy (two or three beam SIM) data can be processed to provide an image exhibiting tight sectioning and high transverse resolution. Our reconstruction algorithm is adapted from the blind-SIM technique which requires very little knowledge of the illumination patterns. It is thus able to deal with illumination distortions induced by the sample or illumination optics. We named this new algorithm thick slice blind-SIM because it models a three-dimensional sample even though only a single two-dimensional plane of focus was measured.

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.

Using spatially non-uniform illumination significantly improves the resolution of light microscopy 1 . Indeed, frequency mixing between the object and the illumination permits the recovery of object frequencies beyond the diffraction-limited detection band pass 2 , 3 , 4 , 5 . However, the image reconstruction process requires a precise knowledge of the illumination patterns (usually focused or periodic) and therefore sophisticated stable mountings 6 , 7 . Here, we show, both theoretically and experimentally, that image reconstruction can be performed without knowing the illumination patterns, provided that their average is roughly homogeneous over the sample. Using blind structured illumination microscopy (blind-SIM), a resolution about two times better than that of conventional wide-field microscopy is obtained by simply illuminating the sample with several uncontrolled random speckles. Our approach is insensitive to specimen or aberration-induced illumination deformations, does not require any calibration step or stringent control of the illumination, and dramatically simplifies the experimental set-up. By illuminating a sample with several uncontrolled random speckles and implementing a blind structured illumination microscopy algorithm, researchers demonstrate that image reconstruction can be achieved without knowing the original illumination pattern, at a resolution two times better than that of conventional wide-field microscopy.

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.

Revealing the molecular orientations of anisotropic materials is desired in materials science and soft-matter physics. Now, an optical diffraction tomographic approach enables the direct reconstruction of dielectric tensors of anisotropic structures in three dimensions.

This article presents a thorough comparison of themain QPM techniques, focusing on their accuracy in terms of measurement precision and trueness. We focus on 8 techniques, namely digital holographic microscopy (DHM), cross-grating wavefront microscopy (CGM), which is based on QLSI (quadriwave lateral shearing interferometry), diffraction phase microscopy (DPM), differential phase-contrast (DPC) microscopy, phase-shifting interferometry (PSI) imaging, Fourier phase microscopy (FPM), spatial light interference microscopy (SLIM), and transport-of-intensity equation (TIE) imaging. For this purpose, we used a home-made numerical toolbox based on discrete dipole approximation (IF-DDA). This toolbox is designed to compute the electromagnetic field at the sample plane of a microscope, irrespective of the object's complexity or the illumination conditions. We upgraded this toolbox to enable it to model any type of QPM, and to take into account shot noise. In a nutshell, the results show that DHM and PSI are inherently free from artefacts and rather suffer from coherent noise; In CGM, DPC, DPM and TIE, there is a trade off between precision and trueness, which can be balanced by varying one experimental parameter; FPM and SLIM suffer from inherent artefacts that cannot be discarded experimentally in most cases, making the techniques not quantitative especially for large objects covering a large part of the field of view, such as eukaryotic cells.

We propose a self-consistent vectorial method, based on a Green's function technique, to describe the Fano resonances that appear in guided mode resonance gratings. The model provides intuitive expressions of the reflectivity and transmittivity matrices of the structure, involving coupling integrals between the modes of a planar reference structure and radiative modes. These expressions are used to derive a physical analysis in configurations where the effect of the incident polarization is not trivial. We provide numerical validations of our model. On a technical point of view, we show how the Green's tensor of our planar reference structure can be expressed as two scalar Green's functions, and how to deal with the singularity of the Green's tensor.

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.