sptPALM

Some of you probably noticed that this blog was down for the last week. Unfortunately our server died right before Thanksgiving and I only now got it back up.  Please let me know if you encounter any problems with it.

To make up for the absence of the blog, I’ll share with you a cool movie we’ve just taken in the NIC. This is single particle tracking PALM or sptPALM [1] of the dopamine receptor labeled with mEos2.  Here, we’re imaging with pretty strong 561 nm illumination and very weak 405 nm illumination, so we’re continuously converting mEos2 molecules to the red state and imaging them until they bleach. Using this methodology we can track many different individual receptors over a long time and see how the population behavior changes when we stimulate the receptor.

References

  1. S. Manley, J.M. Gillette, G.H. Patterson, H. Shroff, H.F. Hess, E. Betzig, and J. Lippincott-Schwartz, "High-density mapping of single-molecule trajectories with photoactivated localization microscopy", Nature Methods, vol. 5, pp. 155-157, 2008. http://dx.doi.org/10.1038/nmeth.1176

2 thoughts on “sptPALM

  1. I recently got an email asking me what laser power we used for this experiment. We used an Agilent laser launch that delivers ~130 mW out of the fiber at 561 nm. We were running at about 20-30% laser power, so ~25-30 mW out of the fiber, assuming the AOTF is linear. In our Nikon TIRF system, the illumination is only about 50% efficient if we measure the laser power out of the objective, so we probably had around 15 mW at the sample. We recently measured the illuminated area on that microscope at 135 um in diameter, so this corresponds to a power density of about 0.1 kW/cm^2, which is right in line with what was reported in Manley et al.

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