Journal: Nature Communications

Article Title: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

doi: 10.1038/s41467-021-24901-3

Figure Lengend Snippet: a Schematic illustration of the SICM-SOFI setup. In SICM, a current is generated through a nanopipette and modulated as a function of the z-position. The image generated reflects a three-dimensional topographical view of the surface at high lateral and axial resolution (top panel). The lateral resolution in SICM ranges from 150 nm to 30 nm and the axial resolution is in the 10 nm range (Supplementary Fig. ). Since the scanning head is mounted on top of the widefield setup, fluorescence imaging can be performed simultaneously. b Conceptual visualization of the SOFI principle. The recorded image stack, containing time traces of independently fluctuating fluorophores is used for analysis. A SOFI image is generated by computing cross-cumulants, creating virtual pixels between adjacent real pixels. The cross-cumulant computation principle can be applied in the axial direction as well if multiple sample planes are simultaneously acquired. For the n th order SOFI image, the cumulant point-spread function volume is raised to n th power thus giving the resolution improvement of a factor of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sqrt{n}$$\end{document} n . The inset images of the actin structure at the bottom of the ( b ) panel represent a standard deviation of the image stack (widefield), 2 nd and 4 th order of linearized SOFI images with corresponding lateral resolution values estimated with an image decorrelation-analysis algorithm of 420 nm, 194 nm, and 93 nm, respectively. c Schematics of correlative SICM and two-color 3D SOFI imaging.

Article Snippet: Fig. 3 Multi-plane 3D SOFI imaging of cytoskeletal components in a fixed COS-7 cell. a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye.

Techniques: Generated, Fluorescence, Imaging, Standard Deviation

Journal: Nature Communications

Article Title: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

doi: 10.1038/s41467-021-24901-3

Figure Lengend Snippet: a Large field of view (100 × 100 μm) 2D SOFI imaging of filamentous actin labeled with a phalloidin-f-HM-SiR self-blinking dye conjugate. b Subsequently, immunostained tubulin labeled with the self-blinking Abberior FLIP-565 dye was imaged. 10 μm x 10 μm zoom-ins of the standard deviation of the image stacks (widefield) and 4 th order SOFI images are shown for a and b panels. 635 nm flat-fielded laser excitation was used for imaging f-HM-SiR and a 561 nm laser line for Abberior FLIP-565. Imaging was performed in a 25% glycerol and PBS mixture at pH = 8. c Corresponding topographical SICM image of the same cell acquired on a dedicated SICM setup. 1024 × 1024 pixels image (78 nm pixel size), acquired at a pixel acquisition rate of 200 Hz with a hopping height of 5 μm. d 3D rendering of a correlative SICM-SOFI overlay. The transparency gradients of tubulin and actin channels are shown as a 2D plane, while topographical SICM information is used for the height representation in the Blender 3D software. The overlay shows that actin is distributed within the boundary of the cell while tubulin displays the canonical cytoskeletal structure. This figure is representative of more than 10 correlative 2D SICM/SOFI single-cell two-color images obtained from at least two independent experiments.

Article Snippet: Fig. 3 Multi-plane 3D SOFI imaging of cytoskeletal components in a fixed COS-7 cell. a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye.

Techniques: Imaging, Labeling, Standard Deviation, Software

Journal: Nature Communications

Article Title: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

doi: 10.1038/s41467-021-24901-3

Figure Lengend Snippet: a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye. A sample volume of 2.45 μm × 65 μm × 55 μm was recorded with 8 equally spaced physical planes, resulting in 22 image planes after 3 rd order SOFI processing. Flat-fielded 635 nm and 532 nm laser lines were used for the excitation. Alexa-647 dye was imaged in reductive/oxidative system imaging buffer with 10 mM mercaptoethylamine, while the Abberior FLIP-565 dye was imaged in a 50% glycerol and PBS solution at pH = 7.5. Height is represented in color scales displayed for each channel. c Co-registered 3D 3 rd order SOFI volumes of tubulin and filamentous actin. Co-registration was performed based on brightfield microscopy images acquired after recording each fluorescence channel. Semi-transparent line shows the cross-sections displayed in Fig. ( a , b ). This figure is representative of more than 10 two-color 3D SOFI images obtained from at least two independent experiments.

Article Snippet: Fig. 3 Multi-plane 3D SOFI imaging of cytoskeletal components in a fixed COS-7 cell. a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye.

Techniques: Imaging, Labeling, Microscopy, Fluorescence

Journal: Nature Communications

Article Title: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

doi: 10.1038/s41467-021-24901-3

Figure Lengend Snippet: Vertical cross-sections of actin ( a ) and tubulin ( b ) in a fixed COS-7 cell marked in Figs. and overlaid with the SICM topography displayed as a semi-transparent contour. Correlative imaging of different cellular components resolved by SICM: filopodia overlaid with fluorescently labeled f-actin from 3D-SOFI ( c ), microvilli overlaid with fluorescently labeled f-actin from 3D-SOFI ( d ) and microtubules of chemically unroofed COS-7 cells overlaid with fluorescently labeled tubulin from 3D-SOFI ( e ). Normalized intensity profiles are displayed below each image. The arrows in c – e are guides for the eye to indicate the locations of cross sections. For overlays only, the intensity values of SOFI images were projected on spatially filtered SICM images for a better structural representation. f Normalized Pearson cross-correlation coefficients of normalized height and intensity cross-sections for both SICM topography and fluorescence channels. Cross-sections ( N = 30) were manually selected (Supplementary Fig. ) from the SICM image for each of the features in 3 different correlative images. Correlation coefficients were measured on filipodia structures, microvilli structures and microtubules. Significantly higher ( p < 0.05, two-sided t -test) correlation values were identified for filopodia(SICM)/actin (2D-SOFI), microvilli (SICM)/actin (3D-SOFI) and microtubules (SICM)/tubulin (3D-SOFI). For all comparisons, p values are shown in the upper part of f panel. The lower and upper bounds of the box plot correspond to the first and third quartiles, while the middle line shows the median. The upper and lower whiskers extend no further than 1.5 times of the interquartile range (IQR) from upper and lower bounds. Points outside this range are defined as outliers. Raw source data are provided in the Source Data File. This figure is representative of more than 10 correlative SICM and 2-color 2D/3D SOFI single-cell images obtained from at least two independent experiments.

Article Snippet: Fig. 3 Multi-plane 3D SOFI imaging of cytoskeletal components in a fixed COS-7 cell. a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye.

Techniques: Imaging, Labeling, Fluorescence

Journal: Nature Communications

Article Title: Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

doi: 10.1038/s41467-021-24901-3

Figure Lengend Snippet: a Topographical SICM map of a fixed COS-7 cell imaged on a dedicated SICM setup after two-color 3D-SOFI acquisition. Corresponding fluorescence images are shown in Fig. . The scan resolution is 1024 × 1024 pixels over an 80 μm × 80 μm area with a corresponding pixel size of 78 nm. The pixel acquisition rate was 200 Hz with a hopping height of 6 μm. Semi-transparent line shows the cross-section area displayed in Fig. ( a , b ). b Leveled zoom-in of the upper part of the cell by mean plane subtraction, showing the topography of microvilli (marked with black arrows) on the surface of the cell. c Spatial band-passed filtered SICM image to highlight multiple microvilli structures. d 3D rendered correlative SICM and two-color 3D-SOFI overlay. The tubulin and actin channels are rendered as volumes consisting of 22 planes, while topographical SICM information is used for a height representation in the Blender 3D software (Supplementary Movie ). e Correlative SOFI and SICM overlay (white arrows correspond to the black arrows in b and c ). This figure is representative of more than 10 correlative SICM and 2-color 3D-SOFI single-cell images obtained from at least two independent experiments.

Article Snippet: Fig. 3 Multi-plane 3D SOFI imaging of cytoskeletal components in a fixed COS-7 cell. a 3D SOFI imaging of filamentous actin labeled with phalloidin conjugated to Alexa-647 and tubulin ( b ) labeled with the Abberior FLIP-565 self-blinking dye.

Techniques: Fluorescence, Software

Journal: Nucleic Acids Research

Article Title: MALISAM: a database of structurally analogous motifs in proteins

doi: 10.1093/nar/gkm698

Figure Lengend Snippet: An analogous pair composed of an interface motif and a core motif. ( a ) The actin filament capping protein CapZ from Gallus gallus (PDB code 1izn). The α subunit is in blue, and the β subunit is in cyan. The three domains in each subunit are represented as a ribbon diagram, a pentagon and a rectangle, respectively. The interface 4-helical bundle motif is in red frame. ( b ) The conserved hypothetical protein Xcc0516 from X. campestris (PDB code 2gsc). The polypeptide chain is colored in rainbow from N-terminus (blue) to C-terminus (orange). The part that is aligned to the hybrid 4-helical bundle motif in CapZ is in red frame. The disordered region between the 2nd and the 3rd helices is represented by a dotted curve. Diagrams are generated by MOLSCRIPT . ( c ) Structure-based sequence alignment of the two 4-helical bundle motifs. The helices in the red frames in ( a ) and ( b ) are diagramed as cylinders in the same colors above or below their corresponding sequences. The PDB code, chain identifier and starting and ending residue numbers are given for each sequence. Columns with two hydrophobic residues are highlighted in yellow.

Article Snippet: An analogous pair composed of an interface motif and a core motif. ( a ) The actin filament capping protein CapZ from Gallus gallus (PDB code 1izn).

Techniques: Generated, Sequencing, Residue