FIG. 7.22 MEMS-in-the-lens arrangement for high numeric aperture confocal microscopy ( Liu et al., 2019b ). More about this image found in MEMS-in-the-lens arrangement for high numeric aperture confocal microscopy ...

FIG. 10.9 (a) Confocal microscopy image of a DPPC monolayer on a flat interface in a Langmuir trough. Contrast in the image is by the preferential segregation of Texas Red DHPE dye to the disordered liquid phase that appears red in the image. The dye is excluded from the semi-crystalline solid phase that appears black. The counterclockwise rotation of the domain arms is due to the chiral center in the native DPPC. The domain sizes are comparable to alveolar dimensions. (b) Compared to the DPPC morphology on a 70 μm diameter bubble. The domains shape changes dramatically to extended interconnected micrometer width stripes. The images were taken at the same surface tension and conditions, the only change is the curvature of the interface. More about this image found in (a) Confocal microscopy image of a DPPC monolayer on a flat interface in a ...

FIG. 3.3 RESOLFT microscopy architecture and comparison with confocal microscopy. (a) Schematic of the RESOLFT microscope. The Gaussian-shaped read-out (or excitation) beam and the doughnut-shaped OFF-switching beam—both having the same wavelength (blue)—are combined with a polarized beam splitter. Successively the two beams are combined with the Gaussian-shaped probe (or ON-switching) beam (magenta) with a dichroic mirror (DM). All co-aligned beams are deflected by the galvanometer mirrors (GMs) and focused by the objective lens (OL) on the sample. Fluorescence (green) is collected by the same objective lens, descanned by the GMs, filtered by the DM, and recorded by a photodetector. (b) Similar to STED microscopy also in RESOLFT microscopy a specific and well-determined temporal beam sequence is necessary to achieve subdiffraction imaging. However, this temporal alignment enables a completely different precision, i.e., microsecond instead of picosecond range. For each sample position (i.e., scanning position or pixel), first the probe beam activates all the fluorescent protein in a well-defined diffraction-limited region, successively the OFF-switching beam de-activates all the fluorescent protein in the periphery of the probed region, leaving only a tiny region close to the center active. Finally, the read-out beam excites this residual region whose fluorescence is registered by the photodetector. This sequence is iterated across the whole sample. Synchronization between the beam temporal sequence, the scanning, and the fluorescence detection can efficiently be implemented by an FPGA-based data acquisition system. (c) Side-by-side comparison of confocal (top) and RESOLFT image of vimentin-rsEGFP2. Scale bar, 1 µm. Adapted from Dreier, J. et al. , Nat. Commun. 10 , 556 (2019). Copyright 2019 Authors. More about this image found in RESOLFT microscopy architecture and comparison with confocal microscopy. (a...

FIG. 9.8 Cells measured using confocal microscopy and dual-arm QME. Representative confocal microscopy of GelMA with (a) ASCs and (d) TAZ activated ASCs with cell nuclei shown in blue and actin filaments in red. (b) OCT and (c) OCT/QME overlay images of ASCs. (e) OCT and (f) OCT/QME overlay images of ASCs with TAZ activation. Cross-sections in the OCT xy and zx planes are represented by orange and purple dashed rectangles, respectively. The arrows indicate example regions where individual cells have elevated elasticity. Scale bars represent 250 µm. Adapted from Hepburn, M. S. et al., Biomed. Opt. Express 11 (2), 867–884 (2020). Copyright 2020 The Optical Society. More about this image found in Cells measured using confocal microscopy and dual-arm QME. Representative c...

FIG. 5.3 Confocal microscopy improves resolution by limiting both the excitation and imaging to select focal spots. (a) The schematic drawing shows the configuration of a conventional confocal microscope that scans a single beam for image generation. (b) Spinning disk confocal microscopy increases... More about this image found in Confocal microscopy improves resolution by limiting both the excitation and...

FIG. 5.11 Calcium imaging using head-fixed mice can capture calcium activity at a variety of spatial resolutions. (a) The schematic drawing shows the setup of the head-fixed mouse imaging. Adapted with permission from Pinto, L. et al., Front. Behav. Neurosci. 12 , 36 ( 2018 ). Copyright 2018 Frontiers Media S.A. (b) Images acquired with confocal microscopy from a head-fixed mouse ( Yoshida et al., 2018 ) at depths of (i) 20 µm and (ii) 127 µm below the cortical surface. Arrowheads indicate putative dendrites in (i) and neuron somas in (ii). Scale bars, 50 µm. Adapted with permission from Yoshida, E. et al., Sci. Rep. 8 (1), 8324 (2018). Copyright 2018 Nature Publishing Group. (c) Calcium activity of mouse acquired with the confocal microscopy. Adapted with permission from Yoshida, E. et al., Sci. Rep. 8 (1), 8324 (2018). Copyright 2018 Nature Publishing Group. More about this image found in Calcium imaging using head-fixed mice can capture calcium activity at a var...

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. (...