# Image Scanning Microscopy to Investigate Polycomb Protein Colocalization onto Chromatin
## 3 Results and Discussion
### Spatial Resolution Improvement by Image Scanning Microscopy  
The optical sectioning ability of a confocal microscope is mainly due to the pinhole spatial filter placed in a conjugate image plane in front of the detector along the fluorescence detection path. Our preliminary measurements (Figure 1) were obtained by setting the pinhole aperture of the confocal system at 1 Airy unit (AU), as commonly performed. In these conditions, the radial (i.e., on the object xy plane) resolution \(d_{xy}\) was 240 nm for the green channel and \(d_{xy}=\) 310 nm for the far-red channel, as judged by the Fourier Ring Correlation (FRC) method [25]. Ideally, by closing the pinhole below 0.2 AU, \(d_{xy}\) would be improved by a factor \(\sqrt{2}\) compared to 1 AU at the price of a dramatic decrease in signal reaching the detector (95%) and severe degradation of the signal to noise ratio (SNR) of the final image. Image Scanning Microscopy (ISM) overcomes this limitation by replacing the single point detector of a confocal microscope with a detector array [28]. Each element of the array generates a \"confocal\" image of the sample, differing in information content as they map different spatial regions of the point spread function generated by the light coming from the objective focus.  
The most transparent way to form an ISM image is represented by the so-called pixel-reassignment (PR) method. To generate the PR-ISM image, all the scanned images are summed together after shifting each image by a vector (shift-vector) proportional to the relative position of the corresponding detector element and properly scaled by a factor. We implemented an adaptive version of the PR method (APR) in which the shift vectors are  
Figure 1: Confocal images of HCC827 nuclei immunolabeled by EZH2/RING1b (upper row), EZH2/BMI1 (**central row**), and EZH2/H3K27 me3 (**lower row**). Scale bar: 10 μm.  
straightforwardly obtained from the data, and there is no need for calibrations or prior information. APR is potentially able to correct misalignment of the system or aberrations introduced by the sample. To extend the resolution further, we also developed alternative approaches fusing together the scanned images by more advanced methods considering also prior knowledge (e.g., the point-spread-functions) of the system and based on inverse filtering or multi-image deconvolution (mode ISM\({}^{++}\)). ISM can be considered the laser-scanning microscopy alternative to wide-field structured illumination microscopy (SIM). In SIM, the sample is illuminated with a series of grating-like light patterns, which encode into the resulting image's normally inaccessible high-frequency information. It is worth noting that the single-photon timing ability of the SPAD array allows the combination of ISM with FLIM, thereby enabling super-resolution FLIM: fluorescence lifetime image-scanning microscopy (FLISM).  
In our study, we used a custom ISM setup whose core is a single-photon avalanche diode (SPAD) array composed of 25 detection elements (organized in a 5 \(\times\) 5 square area configuration) and FPGA-based acquisition electronics. PR-ISM images of PcG proteins and H3K27 me3 appeared qualitatively endowed with higher resolution as compared to their confocal counterparts (Figure 2). Accordingly, FRC analysis showed \(d_{xy}\) = 145 nm (green channel) and \(d_{xy}\) = 210 nm (far-red channel), indicating a resolution improvement of 1.5-1.7\(\times\) over the confocal acquisition. A further significant increase in resolution was obtained by applying the Lucy-Richardson deconvolution to PR-ISM images (Figure 2): ISM\({}^{++}\) images were characterized by \(d_{xy}\) = 95 nm (green channel) and \(d_{xy}\) = 160 nm (far-red channel). These data indicate that ISM may conveniently fit the spatial mesoscale of polycomb domains without the need of special fluorescent labels, sample preparation, or excitation patterns typical of more challenging SRM techniques (e.g., STORM and STED).