Microscopy Club Lecture

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march, 2022

202203mar4:00 PM5:00 PMVirtual EventMicroscopy Club LectureNanometer-scale 3D fluorescence imaging with 4Pi‑STORM 4:00 PM - 5:00 PM OnlineSpeaker:Mark Bates, PhD, Institut für Nanophotonik (IFNANO), Göttingen

Event Details

Chair: Carola Gregor

Speaker: Mark Bates, Institut für Nanophotonik (IFNANO), Göttingen

Nanometer-scale 3D fluorescence imaging with 4Pi‑STORM

Abstract: Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. We report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically, while also using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for localization precision and largely eliminates phase-wrapping artifacts by making optimal use of the information content of the data. With a 3D precision as high as 2 – 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as demonstrated with imaging examples of protein and nucleic acid organization in primary neurons and mammalian mitochondria.

Host: Carola Gregor (MBExC/IFNANO), Jan Huisken (U Göttingen), Eri Sakata (UMG)

Local organizers: Alexander Egner (IFNANO), Jörg Enderlein (U Göttingen)