FIG. 3.1 Image scanning microscopy principles and comparison with confocal images. (a) Schematic of the confocal and image scanning microscopes. Excitation light (blue) passes the dichroic mirror (DM) and is deflected by the galvanometer mirrors (GMs). The pivot point of the scanner is projected by the scan lens (SL) and a tube lens (TL1) into the back aperture of the objective lens (OL). Fluorescence (green) is collected by the OL, descanned by the GMs, filtered by the DM, and projected by a second tube lens (TL2) into the pinhole plane. A telescope or zoom lens is used to image the pinhole plane into the single element detector (confocal microscope) or into an array of detectors (image scanning microscope). Asterisks denote the planes conjugated. (b) Matrix representing the scanned reflection images of a single isolated gold bead (80 nm). Scanned images in the top right and bottom left corners are normalized to the maximum intensity of that image and to the maximum intensity of the central scanned image, respectively. Horizontal and vertical dashed green lines are present to guide the eye. On the right, side-by-side comparison of the PSFs of the ideal confocal (0.2-AU pinhole), the open confocal (1.4-AU pinhole), and ISM. Scale bars, 500 nm. Fingerprint maps superimposed with the estimated shift vectors projected in the image plane are also shown. Scale bars, 100 nm. (c) Side-by-side comparison of ideal confocal, open confocal, and ISM images of tubulin filaments stained with Abberior STAR red. The inset in the ISM image shows the fingerprint map and the estimated shift vectors. Scale bars, 100 nm. Also shown is a composite image including magnified views of the areas outlined by dashed boxes in the main images, together with the ISM image obtained by multi-image deconvolution (ISM++; five iterations). Arrowheads indicate positions used to generate the data in e. Scale bars, 1 µm. (d) Line intensity profiles across two branching tubular filaments at the positions of the arrowheads in c for the different imaging modalities. (e) FRC-based resolution ( Tortarolo et al., 2018 ) as a function of the excitation intensity for the different imaging modalities. Adapted from Castello, M. et al. Nat. Methods 16 (2), 175–178 (2019). Copyright 2019 Authors. More about this image found in Image scanning microscopy principles and comparison with confocal images. (...