No abstract

The aberrations induced by the sample and/or by the sample holder limit the resolution of optical microscopes. Wavefront correction can be achieved using a deformable mirror with wavefront sensorless optimization algorithms but, despite the complexity of these systems, the level of correction is often limited to a small area in the field of view of the microscope.In this work, we present a plug and play module for aberration measurement and correction. The wavefront correction is performed through direct wavefront reconstruction using the spinning-pupil aberration measurement and controlling a deformable lens in closed loop. The lens corrects the aberrations in the center of the field of view, leaving residual aberrations at the margins, that are removed by anisoplanatic deconvolution. We present experimental results obtained in fluorescence microscopy, with a wide field and a light sheet fluorescence microscope. These results indicate that detection and correction over the full field of view can be achieved with a compact transmissive module placed in the detection path of the fluorescence microscope.

Light sheet fluorescence microscopy has become one of the most widely used techniques for three-dimensional imaging due to its high speed and low phototoxicity. Further improvements in 3D microscopy require limiting the light exposure of the sample and increasing the volumetric acquisition rate. We hereby present an imaging technique that allows volumetric reconstruction of the fluorescent sample using spatial modulation on a selective illumination volume. We demonstrate that this can be implemented using an incoherent LED source, avoiding shadowing artifacts, typical of light sheet microscopy. Furthermore, we show that spatial modulation allows the use of Compressive Sensing, reducing the number of modulation patterns to be acquired. We present results on zebrafish embryos which prove that selective spatial modulation can be used to reconstruct relatively large volumes without any mechanical movement. The technique yields an accurate reconstruction of the sample anatomy even at significant compression ratios, achieving higher volumetric acquisition rate and reducing photodamage biological samples.

Structured Illumination Microscopy (SIM) is a key technology for high resolution and super-resolution imaging of biological cells and molecules. The spread of portable and easy-to-align SIM systems requires the development of novel methods to generate a light pattern and to shift it across the field of view of the microscope. Here we show a miniaturized chip that incorporates optical waveguides, splitters, and phase shifters, to generate a 2D structured illumination pattern suitable for SIM microscopy. The chip creates three point-sources, coherent and controlled in phase, without the need for further alignment. Placed in the pupil of a microscope’s objective, the three sources generate a hexagonal illumination pattern on the sample, which is spatially translated thanks to thermal phase shifters. We validate and use the chip, upgrading a commercial inverted fluorescence microscope to a SIM setup and we image biological sample slides, extending the resolution of the microscope.

Optical Projection Tomography (OPT) is a powerful three-dimensional imaging technique used for the observation of millimeter-scaled biological samples, compatible with bright-field and fluorescence contrast. OPT is affected by spatially variant artifacts caused by the fact that light diffraction is not taken into account by the straight-light propagation models used for reconstruction. These artifacts hinder high-resolution imaging with OPT. In this work we show that, by using a multiview imaging approach, a 3D reconstruction of the bright-field contrast can be obtained without the diffraction artifacts typical of OPT, drastically reducing the amount of acquired data, compared to previously reported approaches. The method, purely based on bright-field contrast of the unstained sample, provides a comprehensive picture of the sample anatomy, as demonstrated in vivo on Arabidopsis thaliana and zebrafish embryos. Furthermore, this bright-field reconstruction can be implemented on practically any multi-view light-sheet fluorescence microscope without complex hardware modifications or calibrations, complementing the fluorescence information with tissue anatomy.