α mouse jf549  (Novus Biologicals)

Journal: bioRxiv

Article Title: Multiview super-resolution microscopy

doi: 10.1101/2021.05.21.445200

Figure Lengend Snippet: a) Concept. Diffraction-limited line illumination (blue) is serially introduced into three objectives (OBJ A, B, C) and scanned through the sample, collecting fluorescence (red) from each view. See also Fig. S1. b) 100 nm beads, approximating PSF, as imaged in each view, and fused image after registering/deconvolving all three views. c) Lateral maximum intensity projection image of fixed U2OS cells immunolabeled with mouse-anti-alpha tubulin, anti-mouse-biotin, streptavidin Alexa Fluor 488, marking microtubules (fused result after registration and deconvolution of all three raw views). Height: color bar. d) Raw and fused axial views along the dashed line in c) . Fourier transforms of axial view are shown in last row, showing resolution enhancement after fusion. Orange oval: 1/260 nm −1 in half width and 1/405 nm −1 in half height. e) Lateral (left) and axial (right) maximum intensity projection images of 4x expanded U2OS cell immunolabeled against mitochondrial outer membrane (cyan, rabbit-anti-Tomm20, goat anti-rabbit Biotin, streptavidin Alexa Fluor 488) and double stranded DNA (magenta, mouse anti-dsDNA and donkey anti-Janelia Fluor 549). Triple-view result after registration/deconvolution is shown. f, g) Higher magnification views of yellow and red rectangular regions in e) at indicated axial distance from the beginning of the volume. White arrows: mitochondrial nucleoids encapsulated within Tomm20 signal. h) Axial view corresponding to dashed white line/rectangles in e) highlighting progressive increase in resolution from raw single view (left), to deconvolved single-view (middle), to deconvolved triple-view (right). Red, yellow arrows: mitochondria, nucleoids. See also Movie S1. i) Triple-view reconstruction of whole fixed L1 stage larval worm, nuclei labeled with NucSpot 488. Two maximum intensity projections are shown, taken after rotating volume 220 degrees (top) and 300 degrees (bottom) about x axis. j) Higher magnification lateral view 10 μm from the beginning of the volume, corresponding to the yellow dashed region in i). k) Higher magnification axial views (single planes) corresponding to the red rectangular region in i) , comparing single-view deconvolved result (left) to triple-view result (right). Red arrows highlight subnuclear structure better resolved in triple-view result. Scale bars: b, f-h) 1 μm, c, i) 10 μm, d, e, j, k) 3 μm.

Article Snippet: Kidney tissue sections were incubated with 1μg/mL of mouse-α-Tubulin primary antibody and Goat-α-CD31 primary antibody (R&D Systems, AF3628) in 0.1% Triton-X/PBS at 4°C overnight, washed in 0.1% Triton-X/PBS for 1 hour at RT, and incubated with 1μg/mL of α-mouse-JF549, α-goat-AF647 (Jackson ImmunoResearch, 705-605-147), 10μM of phalloidin-Alexa Fluor 488, and 1μg/mL of DAPI (Thermo Fisher Scientific, D1306) in 0.1% Triton-X/PBS at 4°C overnight.

Techniques: Fluorescence, Immunolabeling, Membrane, Labeling

Journal: bioRxiv

Article Title: Multiview super-resolution microscopy

doi: 10.1101/2021.05.21.445200

Figure Lengend Snippet: a) Triple-view reconstruction of whole fixed adult C. elegans , labeled with NucSpot Live 488. Axial maximum intensity projection is shown. b-e) Comparative higher magnification views of dashed yellow rectangular region in a) , with bottom deconvolved view b) , commercial Leica SP8 confocal microscope c) , conventional triple-view deconvolution d) , attenuation-compensated triple-view deconvolution e) . Colored arrows highlight comparisons, orange: single-vs. triple-view, magenta: deconvolution methods. f) Triple-view reconstruction (green) with segmented nuclei overlaid in red (red), corresponding to red dashed rectangular region in a) . See also Movie S2. g) Schematic of larval wing disc, lateral (top) and axial (bottom) views, including adult muscle precursor myoblasts and notum. h) Lateral plane from triple-view reconstruction, 30 μm from sample surface. Notum nuclei (NLS-mCherry, magenta) and myoblast membranes (CD2-GFP, cyan) labeled. i) Axial maximum intensity projection derived from 6 μm thick yellow rectangle in h). j, k) Higher magnification view of white dashed line/rectangle in h/i) , comparing triple-view result j) to single View C deconvolution k) . White arrows: membrane observed in j) but absent in k). l) Schematic of kidney: approximate region where tissue was extracted. m) Four color triple-view reconstruction of mouse kidney slice. Lateral image at indicated axial height from beginning of the volume, highlighting glomerulus surrounded by convoluted tubules. Red: nuclei stained with DAPI, green: actin stained with phalloidin-Alexa Fluor 488; magenta: tubulin immunolabeled with mouse-α-Tubulin primary, α-Mouse-JF549 secondary; yellow: CD31 immunolabeled with Goat-α-CD31 primary, α-Goat AF647 secondary. n, o) Higher magnification of white rectangular region in m) at indicated axial distance; triple-view reconstruction n) vs. single view C o). p, q) Axial view along dashed line in m) , comparing triple-view reconstruction p) to single view C q) . White arrows: structures that are dim in single view but restored in triple-view. Scale bars: a) 50 μm, b-e, n-q) 10 μm, h, i, m) 20 μm, j, k) 5 μm.

Article Snippet: Kidney tissue sections were incubated with 1μg/mL of mouse-α-Tubulin primary antibody and Goat-α-CD31 primary antibody (R&D Systems, AF3628) in 0.1% Triton-X/PBS at 4°C overnight, washed in 0.1% Triton-X/PBS for 1 hour at RT, and incubated with 1μg/mL of α-mouse-JF549, α-goat-AF647 (Jackson ImmunoResearch, 705-605-147), 10μM of phalloidin-Alexa Fluor 488, and 1μg/mL of DAPI (Thermo Fisher Scientific, D1306) in 0.1% Triton-X/PBS at 4°C overnight.

Techniques: Labeling, Microscopy, Derivative Assay, Membrane, Staining, Immunolabeling