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Event Type:  Seminar (This is an NIH Science event)
Annual Lecture:  Other
Title:  4Pi-RESOLFT Nanoscopy: Nanometer Scale 3D Fluorescence Imaging in Whole Living Cells
Description:  Summary:
By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous images, the signal has to be discriminated against contributions from lobes above and below the focal plane, which has tentatively limited 4Pi arrangements to imaging samples with controllable optical conditions. Here I apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of reversibly switchable fluorescent proteins (RSFPs). I show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. This method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. This approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution. In conclusion, by realizing 4Pi-RESOLFT nanoscopy based on RSFPs, I demonstrate exceptional optical sectioning in coordinate-targeted far-field fluorescence nanoscopy, which greatly facilitates nanometer scale 3D fluorescence imaging in living cells.
superresolution microscopy, nanoscopy, 4pi microscfluorescent proteins (RSFP)
Key References:
Boehm, U., S. W. Hell, & R. Schmidt. (2016) 4Pi-RESOLFT nanoscopy. Nature Communications 7 (10504): 1–8.
Hell, S. W., R. Schmidt, and S. W. Hell (2009) Nature Photonics 3, 381–387.
Schmidt, R., C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner & S. W. Hell (2008) Nature Methods 5, 539–544.
Hell, S. W. & Wichmann, J. (1994) Opt. Lett. 19, 780 – 782.
Hell, S. W. & Stelzer, E. H. K. (1992) Opt. Commun. 93, 277 – 282.
Series Name:  Light Microscopy Interest Group Seminar
Videocast:  Event will not be videocast
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Tuesday, December 12, 2017   11:00am - 12:00pm Add To Outlook Calendar     Add To iCal Calendar     Add To Entourage Calendar
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Name:   Ulrike Boehm
Title:   Dr
Organization:   NIH/NCI
City/Province:   MD
State:   Maryland
Country:   USA

Organization(s):  [NIH] Light Microscopy Interest Group

Location:  On the main NIH Campus
Building:  37
Room:  4107/4041
Street Address:  37 Convent Drive
City:  Bethesda
State:  Maryland
Zip Code:  20892

Name:   Tatiana Karpova
Phone:   240-760-6637
Fax:   240-541-4450
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