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B3 Bayesian Analysis of Blinking and Bleaching

The method is described in the paper "Bayesian localisation microscopy reveals nanoscale podosome dynamics", which will appear in Nature Methods. Bayesian analysis of blinking and bleaching, or B3 microscopy, is a method which analyses data in which many overlapping fluorophores undergo bleaching and blinking events, giving the structure at enhanced resolution. By using a Hidden Markov Model (HMM), it allows useful information to be obtained from data that would be impossible to analyse with standard localisation analysis techniques. Links:

The videos for the paper are below.

Supplemental video 1: Raw data video of vinculin in fixed podosome samples. Samples were labeled with Alexa 488 and mounted in PBS with 100 mM mercaptoethanol added as a reducing agent to induce blinking. Sample was illuminated using a laser at 488nm with a nominal power of 1 kW/cm² . A series of 300 images were collected, taken at 50 frames per second. Scalebar is 500 nm.

Supplemental video 2: Raw data video of live THP-1 cells stably expressing an mCherry-tagged, truncated talin construct. Sample was illuminated with a Xenon arc lamp in the wavelength range 615-687 nm. Video shows the first 500 of 8,000 images taken at frame rates of 50 fps. Scalebar is 2 µm.

Supplemental video 3: Widefield (left) and 3B (right) video of a podosome being dissociated by unwinding. A truncated talin construct is labeled. This video corresponds to Figure 3 (a) in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37°C during imaging. Scalebar is 1 µm.

Supplemental video 4: Widefield (left) and 3B (right) video of a podosome being dissociated by being drawn into its center. A truncated talin construct is labeled. This video corresponds to Figure 3 (b) in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37°C during imaging. Scalebar is 1 µm.

Supplemental video 5: Widefield (left) and 3B (right) video of podosomes being constructed. A truncated talin construct is labeled. This video corresponds to Figure 3 (c) in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37°C during imaging. Scalebar is 1 µm.

Supplemental video 6: Widefield (left) and 3B (right) video of a steady state podosome in which a truncated talin construct is labeled. This video corresponds to Figure 3 (d) in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 50 frames (1 second) apart. Widefield images are created by averaging. Cells were maintained at 37°C during imaging. Scalebar is 1 µm.

Supplemental video 7: Widefield (left) and 3B (right) video reveals podosomes in a motile cell to be highly dynamic. A truncated talin construct is labeled in these cells. This video includes, as a small part, the areas shown in Figure 4 (a)--(c) in the main text. Each widefield and 3B image is generated from 200 frames (4 seconds) of raw data, and are spaced 100 frames (2 seconds) apart. Widefield images are created by averaging. Cells were maintained at 37°C during imaging. Scalebar is 2 µm.

Updated May 11^th 2017, 17:20