Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a Fluorescence image of a typical NIH3T3 cells transfected with Dendra2-Actin plasmid DNA. b A small section of the cell is imaged using scanSMLM and 3D super-resolved volume is reconstructed, where colormap represents the depth. c Few 3D sections are displayed that show Actin filaments at different depths in a single cell. d , e Volume views along with the diagonal views (along lines Q1, Q2 and Q3) showing the arrangement of single Dendra2-Actin molecules. Scale = 1 μm. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a A high-resolution fluorescence image of mitochondria in a NIH3T3 cell (transfected with mEos-Tom20 plasmid DNA). b A section of the mitochondrial network is super-resolved and the volume is reconstructed. The depth is represented by colormap (see colorbar scale). c Few chosen single mitochondria (R1, R2, R3) are resolved that shows the distribution of single molecules (Meos-Tom20). d The corresponding line intensity plots suggests the distribution of single molecules on mitochondria (L1, L2, L3). e , f Volume views and the corresponding diagonal views (along lines, Q1, Q2, Q3) display the organization of single molecules across cell depths in a single mitochondria. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a Cluster map of the distributed HA molecules in a transfected NIH3T3 cell (of few chosen planes at 500 nm, 2500 nm and 4500 nm depth) for both cyclic and conventional scanning. DBSCAN algorithm is used to analyze clusters (with parameters radius ϵ = 114 nm and minimum molecules of 35 for it to be designated as a cluster). Analysis show aggregated HA molecules throughout the cell volume. b Volume image of a cell displaying 3D distribution of HA molecules. In addition, diagonal sectional view of the 3D cluster indicates the local density of clusters across the cell volume. Few enlarged section of a single cluster are also displayed along with orthogonal sectional views (along XY, YZ, XZ) of a specific region. Corresponding intensity plots indicate average HA cluster size of ≈ 240 nm (see, orange arrow) and typical single molecule size (localization precision) of 26.2 nm (see, blue arrow). c Estimated biophysical parameters (average cluster area, density and # molecules per cluster) related to local cell physiology indicates that most of the clusters are spread over a distance of 200 nm with an average density of about 1000#mol./μm 2 . In addition, estimated from the cell volume shows the clustered molecule fraction of ~42.77, indicating that 42.77% of the total HA molecules are clustered, and rest remains unclustered, post 24 hrs of transfection. The clustered area fraction (which is the ratio of clustered area to the total cell area) is found be about 0.981%, indicating a < 1% area is occupied by clusters. These parameters are critical for understanding HA dynamics and help determine underlying biophysical processes. More details can be found in Supplementary note and Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Transfection

Journal: Communications Biology

Article Title: Scanning single molecule localization microscopy (scanSMLM) for super-resolution volume imaging

doi: 10.1038/s42003-023-05364-2

Figure Lengend Snippet: a A cartoon of 3D cell along with real fluorescence image of the transfected NIH3T3 cell. b Cyclic scanning for entire cell volume along with reconstructed super-resolved images ( P 1 to P M ). c Super-resolved images of a few sample planes (plane 1, plane 5, and plane 10) reconstructed from raw recorded data acquired using cyclic and conventional scanning scheme. d The corresponding mean localization precision of all the reconstructed images (plane 1–10). e Plane-wise photobleaching study showing the decrease of fluorescence with time for both cyclic and conventional scan. f Signal-to-background ratio (SBR) for cyclic and conventional scan. g Change in SBR during cyclic scan. h The change in localization density with time. i Amplitude ratio versus time for cyclic scan. Scale = 1 μm. Additional details can be found in Supplementary note .

Article Snippet: NIH3T3 mouse fibroblast cell line is a generous gift from Prof Deepak Nair (Centre for Neuroscience, Indian Institute of Science, Bangalore, India).

Techniques: Fluorescence, Transfection