3D and Multicolor Live-cell Super-resolution Microscopy for Cell Biological Research
Prof. Jörg Bewersdorf, Yale University School of Medicine
Cell biological research relies heavily on the capabilities of light microscopes to resolve structures and processes of interest but is often limited by the spatial (and temporal) resolution of conventional light microscopy . Optical Nanoscopy (super-resolution) techniques such as STED and FPALM/PALM/STORM (single-molecule switching, SMS) microscopy utilize either targeted or stochastic switching of fluorescent molecules to achieve ~25 nm spatial resolution – about 10-fold below the diffraction limit.  The last years have seen many improvements that make these technologies suitable for a rapidly expanding range of applications.
I will report about two recent breakthroughs that significantly expand the live-cell and 3D imaging capabilities of these techniques:
- Optimized instrumentation and labeling approaches now allow multicolor live-cell STED microscopy as a general imaging technique and allow the observation of morphological changes on the ~50-nm scale .
- A new SMS nanoscope using two opposing objectives (4Pi-SMS/iPALM) which takes advantage of adaptive optics and novel data analysis approaches and allows whole-cell imaging at sub-20-nm resolution in 3D .
Joerg Bewersdorf has financial interest in Hamamatsu and Bruker.
 D. Baddeley, J. Bewersdorf “Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images”, Annu. Rev. Biochem 2018, in press
 F. Bottanelli, E.B. Kromann, E.S. Allgeyer, R.S. Erdmann, S. Wood Baguley, G. Sirinakis, A. Schepartz, D. Baddeley, D.K. Toomre, J.E. Rothman, J. Bewersdorf “Two-colour live-cell nanoscale imaging of intracellular targets”, Nature Communication 2016
 F. Huang, G. Sirinakis, E.S. Allgeyer, L.K. Schroeder, W.C. Duim, E.B. Kromann, T. Phan, F.E. Rivera-Molina, J.R. Myers, I. Irnov, M. Lessard, Y. Zhang, M.A. Handel, C. Jacobs-Wagner, C.P. Lusk, J.E. Rothman, D.K. Toomre, M.J. Booth, J. Bewersdorf “Ultra-high resolution 3D imaging of whole cells”, Cell 2016