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.

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.

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.

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.

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.

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.

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.

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.

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.

Improving the axial and lateral resolution of three-dimensional fluorescence microscopy using random speckle illuminations.

We consider a fluorescence microscope in which several three-dimensional images of a sample are recorded for different speckle illuminations. We show, on synthetic data, that by summing the positive deconvolution of each speckle image, one obtains a sample reconstruction with axial and transverse resolutions that compare favorably to that of an ideal confocal microscope.

Superresolution with full-polarized tomographic diffractive microscopy.

Tomographic diffractive microscopy is a three-dimensional imaging technique that reconstructs the permittivity map of the probed sample from its scattered field, measured both in phase and in amplitude. Here, we detail how polarization-resolved measurements permit us to significantly improve the accuracy and the resolution of the reconstructions, compared to the conventional scalar treatments used so far. An isotropic transverse resolution of about 100 nm at a wavelength of 475 nm is demonstrated using this approach.

Optical phase conjugation (OPC)-assisted isotropic focusing.

Isotropic optical focusing - the focusing of light with axial confinement that matches its lateral confinement, is important for a broad range of applications. Conventionally, such focusing is achieved by overlapping the focused beams from a pair of opposite-facing microscope objective lenses. However the exacting requirements for the alignment of the objective lenses and the method's relative intolerance to sample turbidity have significantly limited its utility. In this paper, we present an optical phase conjugation (OPC)-assisted isotropic focusing method that can address both challenges. We exploit the time-reversal nature of OPC playback to naturally guarantee the overlap of the two focused beams even when the objective lenses are significantly misaligned (up to 140 microns transversely and 80 microns axially demonstrated). The scattering correction capability of OPC also enabled us to accomplish isotropic focusing through thick scattering samples (demonstrated with samples of ~7 scattering mean free paths). This method can potentially improve 4Pi microscopy and 3D microstructure patterning.

High-resolution tomographic diffractive microscopy in reflection configuration.

Tomographic diffractive microscopy (TDM) is a label-free imaging technique that reconstructs the 3D refractive index map of the probed object with an improved resolution compared to confocal microscopy. In this work, we consider a TDM implementation in which the sample is deposited on a reflective substrate. We show that this configuration requires calibration and inversion procedures that account for the presence of the substrate for getting highly resolved quantitative reconstructions.

Two-photon fluorescence isotropic-single-objective microscopy.

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.