Bayesian localization microscopy reveals nanoscale podosome dynamics

Abstract

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

Figure 1

Correlative measurements using PALM imaging and Bayesian localization imaging on tubulin. (a, b) Widefield images created by averaging all frames of the PALM image dataset of tubulin-PAGFP (a), and the Bayesian localization image dataset of tubulin-mCherry (b). (c-d) Super-resolution images generated by analyzing the PALM tubulin-PAGFP dataset from a using standard PALM analysis (c) and analyzing the tubulin-mCherry dataset from b using 3B analysis (d). (e) Image generated from the 3B tubulin-mCherry dataset from a using 3B analysis. (f) Overlay of c and e. Green arrows indicate regions with differences in apparent structure that arise from labeling differences. Linescans corresponding to lines (i-iii) are shown in panels (g-i) respectively, with 3B data shown in blue, PALM data shown in pink, 3B PALM shown in green, and widefield data shown in black. Scale bars are 1 μm.

Figure 2

3B analysis of vinculin in fixed cells with podosomes, labeled with Alexa 488. (a) An example maximum likelihood estimate for one set of MCMC samples superimposed on a widefield image created by averaging all 300 images. (b) Probability map created by building up MAP positions created using different sets of MCMC samples. Scale bars correspond to 500 nm. (c,d) Whole cell (widefield and 3B analysis respectively) with areas displayed in (e),(f) and (g) identified by green, turquoise and white rectangles respectively. Scale bars correspond to (a,b) 500 nm, (c,d) 2 μm, (e,f,g) 500 nm.

Figure 3

Podosomes with an mCherry-tagged truncated talin construct forming and dissociating in a live cell. (a, b) A podosome being dissociated (scalebar 400 nm). (c) Podosomes being formed (scalebar 1 μm). (d) A steady state podosome (scalebar 400 nm). Each reconstructed frame uses 200 frames (4 s) and frames are spaced 600 frames (12 s) apart. Videos of the podosomes shown in (a)–(d) are given in Supplementary Videos 3-6 respectively.

Figure 4

Dissociation and formation of groups of podosomes in a motile cell. (a,b) Dissociation and formation of linked podosomes. (c) Separated podosomes joining together. A video containing (a)–(c) along with the rest of the cell is given in Supplementary Video 7. Each reconstructed frame uses 200 frames (4 seconds) and frames are spaced 1000 frames (20 seconds) apart. Scalebars are 800nm.

Figure 5

3B analysis of fixed cell data to determine colocalisation of vinculin and the truncated talin construct in podosomes. (a) Widefield image of vinculin labelled with Alexa-488. (b,c) The individual 3B analysis images shown in glowscale for talin (b) and vinculin (c). (d) Superposition of images from 3B analysis showing the truncated talin construct (in cyan) and vinculin 3B data (in magenta). Scalebars are 1 μm.

Figure 6

Simulations demonstrating the performance of the 3B analysis method. Ground truth simulated image data (a,b), 3B analysis reconstructions (c,d) and linescans (i,j) show the 3B analysis method achieving good reproduction of the structure and 50 nm resolution. For the simulations (a,b), the simulated widefield image (e,f) and a typical frame (g,h) is given. Note that (a,c) correspond to the boxed region in (e,g). Scalebars are 50 nm in (a,c) and 200 nm in all other panels.