Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

doi: 10.1073/pnas.1901176116

Figure Lengend Snippet: Myosin Va teams transport intracellular cargo through networks of actin. (A) Lipid-bound cargo is produced and packaged at the interior of the cell within the Golgi. These cargos are first transported along microtubule tracks, followed by handoff to MyoVa for distribution and final delivery to sites of secretion at the cell membrane. (B) Superresolution, 3D STORM reconstruction of an in vitro actin network. Actin filaments are strung between silica beads of varying diameters, which support the network and maintain a 3D organization. Color represents z position. (Scale bar: 2 µm.) (C) Overlay of 350-nm vesicle trajectory (magenta) by teams of MyoVa within a 3D actin filament network (colored by z position). (Scale bar: 1 µm.)

Article Snippet: 3D STORM images were acquired using a Nikon N-STORM superresolution microscope system with excitation of Alexa-647 phalloidin-labeled actin by 647- and 405-nm lasers.

Techniques: Produced, In Vitro

Journal: Molecular Biology of the Cell

Article Title: Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

doi: 10.1091/mbc.E21-01-0044

Figure Lengend Snippet: Multicolor superresolution observation for frSkylan-S-CLTA with immunostained α-tubulin. (A) Multicolor superresolution image of frSkylan-S-CLTA with immunostained α-tubulin. Cells stably expressing frSkylan-S-CLTA were fixed with 4% PFA/2% GA for 20 min, immunostained by an anti–α-tubulin antibody, and visualized by an Alexa 647–labeled anti-mouse (Fab) 2 antibody. Scale bar, 2 μm. (B) Quantification of the diameters of the CLTA clusters ( n = 6) and the FWHM values of the microtubules by α-tubulin staining ( n = 30), visualized by the multicolor superresolution observation shown in panel A. Bars indicate the mean ± SD.

Article Snippet: An N-STORM (Nikon) superresolution microscope equipped with a 100×/1.49 objective lens (Apo TIRF 100× Oil DIC N2; Nikon) and an EMCCD camera (iXon Du-897; ANDOR) was used to obtain 100,000 images of 256 × 256 pixels (40.96 × 40.96 μm), with a 16 ms exposure time.

Techniques: Stable Transfection, Expressing, Labeling, Staining

Journal: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society

Article Title: Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

doi: 10.1369/0022155412459856

Figure Lengend Snippet: Figure 4. (A) Super-resolution light microscopy image of somatostatin labeling with streptavidin 525-nm quantum dots (QDs) in a 500- nm semithin epoxy section acquired in 3D-SIM mode. The 100-nm resolution of this microscope resolved the irregular distribution of somatostatin in individual secretory granules. Labeling can be seen to be variable with areas of high and moderate fluorescence (arrows). Intensely stained 500- to 600-nm ring-shaped or “doughnut-like” structures in the cytoplasm (square box) and some nonspecific staining in nuclei (colored blue) may also be seen. Super-resolution imaging of QD probes is possible due to their high photostability and strong fluorescence signal. Objective was ×100 with oil immersion and excitation was with a 488-nm laser (monochrome image with false color). (B) Transmission electron microscopy image showing 500- to 600-nm sized granules with electron-dense material at their margins (arrow) thought to correspond to ring-shaped fluorescent structures. (C) Higher power view of a 500-nm secretory granule with electron-dense material inside the limiting membrane (arrows). Scale bars: A = 8 µm, B = 6 µm, C = 500 nm.

Article Snippet: Superresolution fluorescence microscopy in the present study was performed using a Nikon Ni Eclipse inverted microscope fitted with an N-SIM (Structured Illumination Microscopy) illuminator (Nikon Corporation; Tokyo, Japan).

Techniques: Light Microscopy, Labeling, Microscopy, Fluorescence, Staining, Imaging, Transmission Assay, Electron Microscopy, Membrane

Journal: Journal for immunotherapy of cancer

Article Title: Single-chain variable fragment affinity tuning can optimize anti-AML CAR-NK cell functionality.

doi: 10.1136/jitc-2024-010763

Figure Lengend Snippet: Figure 5 Higher affinity 7G3 CAR-NK cells demonstrate increased clustering and cytotoxicity in a short-term time lapse. (A) Representative high-resolution confocal images of the immunological synapse formed between primary CAR-NK cells (untransduced (UTD) or expressing 7G3WT or 7G3L CARs) and GFP-tagged MOLM13 target cells (green). Phalloidin-555 was used to stain actin (red) and Pacific Blue was used to stain cytotoxic perforin granules (blue). Images were captured with a Leica Spinning Disk Confocal microscope at 60X in oil. (B–E) Representative final images from a 30 min time lapse demonstrating LysoTracker blue-stained NK cells (blue) (B) and DRAQ7-stained dead cells (white) (D) after co-culture with MOLM13 target cells, with quantification of normalized mean gray value for each channel (C, E). MOLM13 target cells without NK cell co-culture (No NK) were included as an additional control. Each dashed line represents an individual donor with the mean value from n=3 donors depicted by the solid curve. (F–I) Mean speed (F), maximum speed (G), total distance (H), and maximum distance (I) traveled by NK cells from each treatment group after co-culture with MOLM13 target cells following a 30 min time lapse, as determined through single cell tracking. Individual dots represent individual NK cells from pooled experiments with n=3 donors. Images were captured with a Nikon Eclipse Ti2-E fully motorized inverted microscope with a Yokogawa CSU-W1 SoRa super- resolution spinning disk confocal and 20×/0.45 s Plan Fluor objective. All imaging analysis was performed in Fiji Distribution of ImageJ software47 with use of the TrackMate plugin48 for single cell tracking experiments (C). Detailed statistical data are provided in online supplemental table 2. ****p<0.0001.

Article Snippet: Images were captured with a Nikon Eclipse Ti2- E fully motorized inverted microscope with a Yokogawa CSU- W1 SoRa superresolution spinning disk confocal and 20×/0.45 s Plan Fluor objective.

Techniques: Expressing, Staining, Microscopy, Co-Culture Assay, Control, Single Cell Tracking, Inverted Microscopy, Imaging