Live cell imaging at the nanoscale
Tid: Må 2018-10-15 kl 13.15 - 14.00
Föreläsare: Ilaria Testa, Institutionen för tillämpad fysik, KTH
Plats: Obs, ny lokal! Sal FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21,Stockholm
Presentationen kommer att hållas på engelska.
Lens-based microscopy was unable to discern fluorescently labeled features closer than 200 nm for decades, until the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off quickly made nanoscale imaging routine. Reported nanoscopy variants switch these features either in a target manner with intense laser beams, or molecule by molecule followed by computation in a stochastic fashion. Here, we show that emergent MoNaLISA fluorescence nanoscopy enables fast and continuous imaging of living cells and tissues in super resolved detail by producing raw data images using only ultralow levels of light. This advance has been facilitated by the generation of fluorescent proteins (rsFP) that can be reversibly photoswitched numerous times. Distributions of functional rsFP-fusion proteins in living bacteria and mammalian cells are imaged at < 40 nm resolution. Using a fast-switching rsFP variant, we increased the imaging speed over our first reported RESOLFT schemes, which in turn enabled us to record spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours. Furthermore, 3D and adaptive scanning implementation of our concept enable precise localization of synapses by recording neuronal proteins located in the pre and post synaptic side as well as in the axon initial segment directly inside 3D tissues. Our powerful next generation super resolution technique represents a new paradigm for non invasive induction and monitoring of ultrastructural dynamics of synaptic plasticity at the nanoscale.