Advances in Optical Nanoscopy of Living Cells

The diffraction limit of light has constrained the resolution of light microscopes in the far field since its discovery more than a century ago. Structures smaller than about half the wavelength of light could therefore not be resolved by light microscopes. The realization that this limit can be broken has triggered a revolution in imaging, especially in biological applications which heavily depend on light microscopy. About 25 nm spatial resolution and beyond, more than 10-fold better than in conventional microscopy, can now be achieved [1].
 
In this presentation, I will provide an overview over current developments in my group in STED and single molecule switching (SMS) nanoscopy (e.g. FPALM, PALM or STORM), two of the most prominent nanoscopy techniques. I will present the combination of STED microscopy with total internal reflection (TIRF) microscopy which limits excitation to a vicinity of ~100 nm to the cover slip and reduces bleaching and photo damage in live cell imaging [2].
 
Furthermore, I will describe our latest results in 3D STED microscopy of scattering specimens enabled by the integration of adaptive optics into a custom STED microscope [3]. I will especially focus on our latest achievement in SMS nanoscopy; we have recently improved the recording speed dramatically by developing new algorithms which allow the use of new sCMOS cameras for quantitative imaging [4]. In combination with multi-emitter fitting algorithms, we have achieved SMS nanoscopy of live cells at speeds ranging from 0.5 to 30 frames per seconds.

Speaker: Joerg Bewersdorf, Yale University