Luke Lavis Seminar

Last week, we were fortunate to have Luke Lavis, from Janelia Farm, visit UCSF for a seminar. The Lavis lab develops novel fluorophores for microscopy applications, including new fluorophores for super-resolution imaging. I’ll summarize some of the highlights of his seminar below.

Fluorophore structures

Fluorophore structures. Figure from Luke Lavis.


He first discussed the development of caged fluorophores for super-resolution imaging. These are dye molecules that are rendered non-fluorescent by a UV-cleavable caging group. His lab has developed novel strategies to prepare caged fluoresceins and rhodamines [1]. These can be used as straightforward probes for super-resolution imaging by single-molecule switching and localizaiton microscopy. UV illumination removes the cage, and then the fluorescent dye can be imaged until it bleaches. Of the dyes reported in this paper the red rhodamine Q and green rhodamine 110 have the best performance, with brightness (assessed by the number of photons recorded from a single molecule) comparable to mEos2. These are conceptually similar to the caged dyes sold by Abberior, but have different structures and different caging groups.

More recently, his group has prepared analogs of fluorescein and rhodamine where the xanthene oxygen is replaced with a carbon, prodcuing carbofluorescein and carborhodamine. Both carbofluorescein and carborhodamine are red dyes, with absorption around 545 nm and emission around 565 nm. Caged carborhodamine 110 makes an excellent probe for super-resolution imaging, as it is brighter than either mEos2 or Cy5. [2]

Finally, his group has also prepared caged fluorophores that are uncagable not by light but by enzymatic cleavage.  They have then engineered caging groups that are not recognized by any mammalian enzymes but are recognized by a specific enzyme that can be introduced into cells. Transgenic cells can be specifically labeled by incubation with the caged dye, allowing connectivity to be probed by following the diffusion of the uncaged dye.  The same caging groups can be applied to other small molecules, allowing specific delivery of pharmacologic agents only to the transgenic cells. [3]

 

References

  1. L.M. Wysocki, J.B. Grimm, A.N. Tkachuk, T.A. Brown, E. Betzig, and L.D. Lavis, "Facile and General Synthesis of Photoactivatable Xanthene Dyes", Angewandte Chemie International Edition, vol. 50, pp. 11206-11209, 2011. http://dx.doi.org/10.1002/anie.201104571
  2. J.B. Grimm, A.J. Sung, W.R. Legant, P. Hulamm, S.M. Matlosz, E. Betzig, and L.D. Lavis, "Carbofluoresceins and Carborhodamines as Scaffolds for High-Contrast Fluorogenic Probes", ACS Chemical Biology, vol. 8, pp. 1303-1310, 2013. http://dx.doi.org/10.1021/cb4000822
  3. L. Tian, Y. Yang, L.M. Wysocki, A.C. Arnold, A. Hu, B. Ravichandran, S.M. Sternson, L.L. Looger, and L.D. Lavis, "Selective esterase-ester pair for targeting small molecules with cellular specificity", Proceedings of the National Academy of Sciences, vol. 109, pp. 4756-4761, 2012. http://dx.doi.org/10.1073/pnas.1111943109