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BP: Fachverband Biologische Physik

BP 7: Superresolution Optical Microscopy (focus session)

BP 7.8: Vortrag

Montag, 16. März 2015, 16:45–17:00, H 1028

Optimizing STED Performance — •Marcelle Koenig, Rhys Dowler, Benedikt Kraemer, Felix Koberling, Sebastian Tannert, Matthias Patting, and Rainer Erdmann — PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, info@picoquant.com

Stimulated Emisson Depletion (STED) microscopy is becoming a standard technique in biological imaging, reaching an optical resolution far below 100*nm. The improvement in optical resolution can be achieved with different optical tools and data acquisition, as well as data processing workflows. These have a significant influence not only on the optical resolution itself but also on the general applicability of the technique for specific labels, specimen and phenomena to be studied. We present results based on a confocal microscope which was upgraded with an EASYDOnut phaseplate to convert the STED laser beam into the required donut-shaped focal spot while leaving the co-aligned excitation beam unaffected [1]. On the way towards suitable imaging conditions, various experimental modalities to minimize irreversible photobleaching and to improve photon statistics will be discussed. Different analysis methods based upon the arrival times of photons have also been compared in order to sharpen the images and to suppress unwanted contributions in addition to spectral filtering. Multilabel STED with only one depletion wavelength can be achieved by employing spectral as well as temporal information which act as a fingerprint for individual dyes. Pattern Matching analysis allows for a fast and simple separation of the different fluorescent labels.