4d super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues4 d超分辨率显微术与传统荧光团和单波长激发光厚的细胞和组织.pdfVIP

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues 1 1 2 1 1 David Baddeley , David Crossman , Sabrina Rossberger , Juliette E. Cheyne , Johanna M. Montgomery , 1 2 1 1 Isuru D. Jayasinghe , Christoph Cremer , Mark B. Cannell , Christian Soeller * 1 Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand, 2 Kirchhoff-Institute for Physics, University of Heidelberg, Heidelberg, Germany Abstract Background: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. Methodology/Principal Findings: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ,15 nm (std. dev.) while reducing marker cross-talk to ,1%. Using astigmatism an axial resolution of ,65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule event