Quantitative phase microscopies: accuracy comparison.

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.

Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging.

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.

Dry mass photometry of single bacteria using quantitative wavefront microscopy.

Quantitative phase microscopy (QPM) represents a noninvasive alternative to fluorescence microscopy for cell observation with high contrast and for the quantitative measurement of dry mass (DM) and growth rate at the single-cell level. While DM measurements using QPM have been widely conducted on mammalian cells, bacteria have been less investigated, presumably due to the high resolution and high sensitivity required by their smaller size. This article demonstrates the use of cross-grating wavefront microscopy, a high-resolution and high-sensitivity QPM, for accurate DM measurement and monitoring of single microorganisms (bacteria and archaea). The article covers strategies for overcoming light diffraction and sample focusing, and introduces the concepts of normalized optical volume and optical polarizability (OP) to gain additional information beyond DM. The algorithms for DM, optical volume, and OP measurements are illustrated through two case studies: monitoring DM evolution in a microscale colony-forming unit as a function of temperature, and using OP as a potential species-specific signature.

Scalar approximation of Maxwell equations: derivation and accuracy.

Replacing Maxwell equations by a scalar wave equation is often used in computational imaging to simulate the light-sample interaction. It significantly reduces the computational burden but provides field maps that are insensitive to the polarization of the incident field, provided the latter is constant throughout the sample. Here, we develop a scalar approximation that accounts for the polarization of the incident field. Comparisons with rigorous simulations show that this approach is more accurate than the classical scalar approximation with similar computational cost.

3D-coherent anti-Stokes Raman scattering Fourier ptychography tomography (CARS-FPT).

Fourier ptychography tomography (FPT) is a novel computational technique for coherent imaging in which the sample is numerically reconstructed from images acquired under various illumination directions. FPT is able to provide three-dimensional (3D) reconstructions of the complex sample permittivity with an increased resolution compared to standard microscopy. In this work, FPT is applied to coherent anti-Stokes Raman scattering (CARS) imaging. We show on synthetic data that complex third-order susceptibilities can be reconstructed in 3D from a limited number of widefield CARS images. In addition, we observe that the non-linear interaction increases significantly the potential of CARS-FPT compared to linear FPT in terms of resolution. In particular, with a careful choice of the pump and Stokes beam directions, CARS-FPT is able to provide optical sectioning even in transmission configuration.

Super-resolved live-cell imaging using random illumination microscopy.

Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.

Three-dimensional imaging with reflection synthetic confocal microscopy.

Biomedical imaging lacks label-free microscopy techniques able to reconstruct the contour of biological cells in solution, in 3D and with high resolution, as required for the fast diagnosis of numerous diseases. Inspired by computational optical coherence tomography techniques, we present a tomographic diffractive microscope in reflection geometry used as a synthetic confocal microscope, compatible with this goal and validated with the 3D reconstruction of a human effector T lymphocyte.

Inverse scattering for reflection intensity phase microscopy.

Reflection phase imaging provides label-free, high-resolution characterization of biological samples, typically using interferometric-based techniques. Here, we investigate reflection phase microscopy from intensity-only measurements under diverse illumination. We evaluate the forward and inverse scattering model based on the first Born approximation for imaging scattering objects above a glass slide. Under this design, the measured field combines linear forward-scattering and height-dependent nonlinear back-scattering from the object that complicates object phase recovery. Using only the forward-scattering, we derive a linear inverse scattering model and evaluate this model's validity range in simulation and experiment using a standard reflection microscope modified with a programmable light source. Our method provides enhanced contrast of thin, weakly scattering samples that complement transmission techniques. This model provides a promising development for creating simplified intensity-based reflection quantitative phase imaging systems easily adoptable for biological research.

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.

Numerical approach for reducing out-of-focus light in bright-field fluorescence microscopy and superresolution speckle microscopy.

The standard two-dimensional (2D) image recorded in bright-field fluorescence microscopy is rigorously modeled by a convolution process involving a three-dimensional (3D) sample and a 3D point spread function. We show on synthetic and experimental data that deconvolving the 2D image using the appropriate 3D point spread function reduces the contribution of the out-of-focus fluorescence, resulting in a better image contrast and resolution. This approach is particularly interesting for superresolution speckle microscopy, in which the resolution gain stems directly from the efficiency of the deconvolution of each speckle image.

Versatile inversion tool for phaseless optical diffraction tomography.

Estimating three-dimensional complex permittivity of a sample from the intensity recorded at the image plane of a microscope for various angles of illumination, as in optical Fourier ptychography microscopy, permits one to avoid the interferometric measurements of classical tomographic diffraction microscopes (TDMs). In this work, we present a general inversion scheme for processing intensities that can be applied to any microscope configuration (transmission or reflection, low or high numerical aperture), scattering regime (single or multiple scattering), or sample-holder geometries (with or without substrate). The inversion procedure is tested on a wide variety of synthetic experiments, and the reconstructions are compared to that of TDMs. In most cases, phaseless data yield the same result as complex data, thus paving the way toward a drastic simplification of TDM implementation.

Versatile inversion tool for phaseless optical diffraction tomography: publisher's note.

This publisher's note corrects a typo in the title of J. Opt. Soc. Am. A36, C1 (2019)JOAOD60740-323210.1364/JOSAA.36.0000C1.

Coherent anti-Stokes Raman Fourier ptychography.

We present a theoretical and numerical study of coherent anti-Stokes Raman scattering Fourier ptychography microscopy (CARS-FPM), a scheme that has not been considered so far in the previously reported CARS wide-field imaging schemes. In this approach, the distribution of the Raman scatterer density of the sample is reconstructed numerically from CARS images obtained under various angles of incidences of the pump or Stokes beam. Our inversion procedure is based on an accurate vectorial model linking the CARS image to the sample and yields both the real and imaginary parts of the susceptibility, the latter giving access to the Raman information, with an improved resolution.

Quantitative model of the image of a radiating dipole through a microscope.

In this paper, we introduce a formalism to determine the relationship between the full vectorial electric field existing at the object plane of a microscope and that existing at the image plane. The model is then used to quantitatively simulate, in both phase and intensity, the image of a radiating electric dipole placed either in a homogeneous medium or in the vicinity of a substrate. These simulations are compared with experimental measurements on single gold nanoparticles carried out by quadriwave lateral shearing interferometry.

Multi-wavelength multi-angle reflection tomography.

We have developed a reflection tomographic microscope in which the sample is reconstructed from different holograms recorded under various angles and wavelengths of incidence. We present an iterative inversion algorithm based on a rigorous modeling of the wave-sample interaction that processes all the data simultaneously to estimate the sample permittivity distribution. We show that using several wavelengths permits a significant improvement of the reconstruction, especially along the optical axis.

Two-photon speckle illumination for super-resolution microscopy.

We present a numerical study of a microscopy setup in which the sample is illuminated with uncontrolled speckle patterns and the two-photon excitation fluorescence is collected on a camera. We show that, using a simple deconvolution algorithm for processing the speckle low-resolution images, this wide-field imaging technique exhibits resolution significantly better than that of two-photon excitation scanning microscopy or one-photon excitation bright-field microscopy.

Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy.

Total internal reflection microscopy is mainly used in its fluorescence mode and is the reference technique to image fluorescent proteins in the vicinity of cell membranes. Here, we show that this technique can easily become a phase microscope by simply detecting the coherent signal resulting from the interference between the field scattered by the probed sample and the total internal reflection. Moreover, combining several illumination angles permits generating synthetic aperture reconstructions with improved resolutions compared to standard label-free microscopy techniques.

Unified description of three-dimensional optical diffraction microscopy: from transmission microscopy to optical coherence tomography: tutorial.

In this tutorial, we present a general model linking the data provided by any optical diffraction microscope to the sample permittivity. Our analysis is applicable to essentially all microscope configurations, in transmission or reflection mode, using scanning or full-field illumination, with or without interferometric measurements. We include also a generalization of our analysis to vector fields.

Dual fluorescence-absorption deconvolution applied to extended-depth-of-field microscopy.

Fast imaging over large volumes can be obtained in a simple manner with extended-depth-of-field (EDOF) microscopy. A standard technique of Wiener deconvolution can correct for the blurring inherent in EDOF images. We compare Wiener deconvolution with an alternative, parameter-free technique based on the dual reconstruction of fluorescence and absorption layers in a sample. This alternative technique provides significantly enhanced reconstruction contrast owing to a quadratic positivity constraint that intrinsically favors sparse solutions. We demonstrate the advantages of this technique with mouse neuronal images acquired in vivo.