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.

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.

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.

Joint Reconstruction Strategy for Structured Illumination Microscopy With Unknown Illuminations.

The blind structured illumination microscopy strategy proposed by Mudry et al. is fully re-founded in this paper, unveiling the central role of the sparsity of the illumination patterns in the mechanism that drives super-resolution in the method. A numerical analysis shows that the resolving power of the method can be further enhanced with optimized one-photon or two-photon speckle illuminations. A much improved numerical implementation is provided for the reconstruction problem under the image positivity constraint. This algorithm rests on a new preconditioned proximal iteration faster than existing solutions, paving the way to 3D and real-time 2D reconstruction.

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.