Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging

The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an Extended Depth of Field (EDF) approach in a Random Illumination Microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets.