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.

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.

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.

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.

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.

High-resolution total-internal-reflection fluorescence microscopy using periodically nanostructured glass slides.

We compare the performance of a total-internal-reflection fluorescence microscope under varying illumination and substrate conditions. The samples are deposited on a standard homogeneous glass slide or on a grating and illuminated by one or two interfering beams at various incident angles. A conjugate gradient with positivity a priori information is used to reconstruct the fluorophore density from the images. Numerical studies demonstrate that when the sample lies on an optimized grating, the lateral resolution of the microscope is greatly improved, up to fourfold, the best result being obtained when the grating is illuminated by two interfering beams.

Experimental demonstration of quantitative imaging beyond Abbe's limit with optical diffraction tomography.

Optical diffraction tomography (ODT) is a recent imaging technique that combines the experimental methods of phase microscopy and synthetic aperture with the mathematical tools of inverse scattering theory. We show experimentally that this approach permits us to obtain the map of permittivity of highly scattering samples with axial and transverse resolutions that are much better than that of a microscope with the same numerical aperture.

Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate.

We propose a fluorescence surface imaging system that presents a power of resolution beyond that of the diffraction limit without resorting to saturation effects or probe scanning. This is achieved by depositing the sample on an optimized periodically nanostructured substrate in a standard total internal reflection fluorescence microscope. The grating generates a high-spatial-frequency light grid that can be moved throughout the sample by changing the incident angle. An appropriate reconstruction procedure permits one to recover the fluorescence amplitude from the images obtained for various incidences. Simulations of this imaging system show that the resolution is not limited by diffraction but by the period of the grating.

Influence of multiple scattering on the resolution of an imaging system: a Cramér-Rao analysis.

We revisit the notion of resolution of an imaging system in the light of a probabilistic concept, the Cramér-Rao bound (CRB). We show that the CRB provides a simple quantitative estimation of the accuracy one can expect in measuring an unknown parameter from a scattering experiment. We then investigate the influence of multiple scattering on the CRB for the estimation of the interdistance between two objects in a typical two-sphere scattering experiments. We show that, contrarily to a common belief, the occurence of strong multiple scattering does not automatically lead to a resolution enhancement.

Increasing the lateral resolution of 4Pi fluorescence microscopes.

The axial resolution of fluorescence microscopes can be considerably improved by superposing two illumination beams and by adding coherently the two wavefronts emitted by the luminescent sample. This solution has been implemented in 4Pi microscopes. Theoretical and experimental results have shown that a considerable improvement of the axial resolution can be obtained with these microscopes. However, the lateral resolution remains limited by diffraction. We propose a configuration of a 4Pi microscope in which the lateral displacement of the source modifies the collection efficiency function (CEF). Numerical calculations based on an approximate scalar theory and on exact vector-wave-optics results of the field distribution of the electromagnetic field in image space show that the lateral extent of the CEF can be reduced by a factor greater than 2 with respect to the diffraction limit. We show that, with this solution, the resolution in the transverse plane of 4Pi type B and 4Pi type C microscopes can be improved significantly.

Determination of refractive indices of opaque rough surfaces.

The refractive indices of optical materials are usually determined from spectrophotometric and ellipsometric measurements of specular beams. When the roughness of the interfaces increases, the energy in the specularly reflected and transmitted beams decreases and scattering becomes predominant. For strong roughness (compared to the incident wavelength) a surface does not exhibit specular reflection or transmission, making difficult the determination of the refractive index. We describe two techniques, based on scattering measurements, that one can use to determine the refractive indices of opaque inhomogeneous media.

Angle-resolved ellipsometry of light scattering: discrimination of surface and bulk effects in substrates and optical coatings.

Angle-resolved ellipsometry of light scattering is an original technique developed at the Fresnel Institute to identify scattering processes in substrates and multilayers. We extend the investigation because numerous experimental results proved that the technique can be of major interest for analysis of microcomponents and their scattering origins. Surface and bulk effects can be separated in most situations, as well as the oblique growth of materials and the presence of first-order contaminants.