Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

Enhancing fluorescence in vivo imaging using inorganic nanoprobes.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities.

Imagery of local defects in multilayer components by short coherence length interferometry.

We propose to image local defects inside multidielectric optical components by using a special configuration Linnik interference microscope, along with a CCD camera and a dedicated detection, to extract the amplitude scattered by the defects in the interference image. The use of a short coherence length source allows one to obtain interference only from a thin slice (~1microm) within the observed object. The object is tilted to permit the use of a dark-field configuration to enhance the defect contrast. We describe the experimental setup and the detection scheme. Images that exhibit local point defects on the interfaces of various multilayer optical components (laser mirrors) are presented.

Birefringence imaging with imperfect benches: application to large-scale birefringence measurements.

We describe a birefringence measurement bench that allows fast two-dimensional measurements of low-birefringence fields in large transparent samples. We present calculations that show that, even when a birefringence bench exhibits defects (nonideal components, misalignments, etc.), measurements can be performed under realistic conditions without any a priori knowledge of the origin of the bench defects. This allows the measurement of birefringence fields on large-scale samples by use of an array of detectors instead of a single detector element, with a sensitivity of 3 x 10(-4) rad for 2-s data integration.