FLUORECENCE MICROSCOPY SUPERRESOLUTION BLINK MICROSCOPY ON THE BASIS OF ENGINEERED DARK STATES* *Christian Steinhauer, Carsten Forthmann, Jan Vogelsang, and Philip Tinnefeld Angewandte Physik - Biophysik, and Center for NanoScience, Ludwig-Maximilians-Universität, Amalienstrasse 54, 80799 München, Germany Received August 19, 2008; E-mail: Philip.Tinnefeld@lmu.de Presented by Jacqueline Anna Janssen, 27th of May 2013, Munich

MAIN TOPICS INTRODUCTION Fluorescence microscopy, definition and short description Abbe diffraction limit, its consequences, possible ways to solve the problem Conventional microscopy vs. super-resolution microscopy. ON, OFF, Dark states of fluorophores SUPER-RESOLUTION TECHNIQUES, THE USE OF STOCHASTIC READOUT Optimization of the resolution, introducing BM Switching mechanisms: Photoinduced switching mechanisms The Reducing and Oxidizing System (ROXS): Controlling the Fluorescence of Single Fluorophores Scientific paper written by Philip Tinnefeld et al., results of the experiments Application of the method and its importance Sources

Fig. 1: Schematic of a fluorescence microscope.

𝑑= λ 2nsin θ Fig. 2: Comparison of conventional microscopy and super-resolution microscopy.

Fig. 3: Schematic representation of the method based on a simplified Jablonski diagram.

Fig. 4: Possible behavior of the molecule residing on the the excited State S1

Fig. 5: Different possibilities to induce photoinduced blinking in single molecules. a) Photochromic fluorophores can be switched directly via two different wavelengths. b) Triplet states are used as dark states. c) Radical states are used as controllable dark states. d) Fluorophores are switched via a non absorbing and absorbing FRET-acceptor.

Fig. 6: Schematic drawing of photoinduced processes of common organic fluorophores including radical states.

Fig. 7: Concept of the reducing and oxidizing system (ROXS)

Fig. 8: Controlling blinking for superresolution microscopy. (a) Part of a fluorescence transient of single Cy5 with ascorbic acid. (b)Dependence of τoff on AA concentration. (c) Dependence of τon, τoff, and the number of photons detected per on-state (on-counts) on excitation power

ATTO647N addition of AA to oxygen depleted solutions Table 1: Triplet lifetime and radical anion lifetimes in the abundance of the following substances. ATTO565 (6±2) ms to (20±5) ms Cy3B (11±4) ms to (33±12) ms Alexa 647 (13±3) ms to (52±8) ms ATTO647N addition of AA to oxygen depleted solutions remaining at (28±7) ms

Fig. 9: (a) Total internal reflection (left) and “Blink Microscopy” (right) images of actin filaments labeled with Alexa647. (b) Wide-field (left) and superresolution (right) images of microtubule in fixed 3T3 fibroblast labeled with Alexa647-Fab-fragments.

Fig. 10: Total internal reflection fluorescence microscopy of ATTO655-phalloidin-labeled single actin filaments (a and b) and bundled actin filaments in fixed NIH/3T3 cells (d and e)

Fig. 11: Super-resolution microscopy with DNA origami

SOURCES www.pnas.orgcgidoi10.1073pnas.0811875106 Vogelsang et al.; Controlling the fluorescence of ordinary oxazine dyes for single- molecule switching and superresolution microscopy; PNAS May 19, 2009 vol. 106 no. 20, 8107–8112 WIREs Nanomed Nanobiotechnol 2012, 4:66–81. doi: 10.1002/wnan.173 Jan Vogelsang (Dissertation); Advancing Single-Molecule Fluorescence Spectroscopy and Super-Resolution Microscopy with Organic Fluorophores, Munich, September 2009 Tinnefeld et al.; Superresolution Microscopy on the Basis of Engineered Dark States; 16840 9 J. Am. Chem. Soc. 2008, 130, 16840–16841 Tinnefeld et al.; Make them Blink: Probes for Super-Resolution Microscopy; ChemPhysChem 2010, 11, 2475 – 2490 Tinnefeld et al.; Resolving Single-Molecule Assembled Patterns with Superresolution Blink-Microscopy; DOI: 10.1021/nl903730r, Nano Lett. 2010, 10, 645-651