Compressive Image Scanning Microscope

We present a novel approach to implement compressive sensing in laser scanning microscopes (LSM), specifically in image scanning microscopy (ISM), using a single-photon avalanche diode (SPAD) array detector. Our method addresses two significant limitations in applying compressive sensing to LSM: the time to compute the sampling matrix and the quality of reconstructed images. We employ a fixed sampling strategy, skipping alternate rows and columns during data acquisition, which reduces the number of points scanned by a factor of four and eliminates the need to compute different sampling matrices. By exploiting the parallel images generated by the SPAD array, we improve the quality of the reconstructed compressive-ISM images compared to standard compressive confocal LSM images. Our results demonstrate the effectiveness of our approach in producing higher-quality images with reduced data acquisition time and potential benefits in reducing photobleaching.

Reconstructing the Image Scanning Microscopy Dataset: an Inverse Problem

Confocal laser-scanning microscopy (CLSM) is one of the most popular optical architectures for fluorescence imaging. In CLSM, a focused laser beam excites the fluorescence emission from a specific specimen position. Some actuators scan the probed region across the sample and a photodetector collects a single intensity value for each scan point, building a two-dimensional image pixel-by-pixel. Recently, new fast single-photon array detectors have allowed the recording of a full bi-dimensional image of the probed region for each scan point, transforming CLSM into image scanning microscopy (ISM). This latter offers significant improvements over traditional imaging but requires an optimal processing tool to extract a super-resolved image from the four-dimensional dataset. Here we describe the image formation process in ISM from a statistical point of view, and we use the Bayesian framework to formulate a multi-image deconvolution problem. Notably, the single-photon detector suffers exclusively from the photon shot noise, enabling the development of an effective likelihood model. We derive an iterative likelihood maximization algorithm and test it on experimental and simulated data. Furthermore, we demonstrate that the ISM dataset is redundant, enabling the possibility of obtaining reconstruction sampled at twice the scanning step. Our results prove that in ISM, under appropriate conditions, the Nyquist-Shannon sampling criterium is effectively relaxed. This finding can be exploited to speed up the acquisition process by a factor of four, further improving the versatility of ISM systems.