Ultra-stable and versatile widefield cryo-fluorescence microscope for single-molecule localization with sub-nanometer accuracy.

We developed a stand-alone cryostat with optical access to the sample which can be adapted to any epi-fluorescence microscope for single-molecule fluorescence spectroscopy and imaging. The cryostat cools the sample to a cryogenic temperature of 89 K, and allows for imaging single molecules using an air objective with a numerical aperture of 0.7. An important property of this system is its excellent thermal and mechanical stability, enabling long-time observations of samples over several hours with negligible drift. Using this system, we performed photo-bleaching studies of Atto647N dye molecules, and find an improvement of the photostability of these molecules by more than two orders of magnitude. The resulting increased photon numbers of several millions allow for single-molecule localization accuracy of sub-nanometer.

[1]  Ian M. Dobbie,et al.  Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions , 2014, Nano letters.

[2]  Christoph Hagen,et al.  Fluorescence cryo-microscopy: current challenges and prospects , 2014, Current opinion in chemical biology.

[3]  Grant J. Jensen,et al.  Correlated cryogenic photoactivated localization microscopy and electron cryotomography , 2014, Nature Methods.

[4]  Alois Renn,et al.  Cryogenic colocalization microscopy for nanometer-distance measurements. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[5]  Michel Orrit,et al.  Single-molecule photophysics, from cryogenic to ambient conditions. , 2014, Chemical Society reviews.

[6]  C. Hübner,et al.  The spectroscopic ruler revisited at 77 K. , 2013, Physical chemistry chemical physics : PCCP.

[7]  M. Brecht,et al.  Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures. , 2012, The Review of scientific instruments.

[8]  M. Sauer,et al.  rapidSTORM: accurate, fast open-source software for localization microscopy , 2012, Nature Methods.

[9]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[10]  J. Dubochet,et al.  Cryo‐EM—the first thirty years , 2012, Journal of microscopy.

[11]  John A.G. Briggs,et al.  Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision , 2011, The Journal of cell biology.

[12]  C. Hübner,et al.  A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K. , 2010, The Review of scientific instruments.

[13]  Felix J. B. Bäuerlein,et al.  Micromachining tools and correlative approaches for cellular cryo-electron tomography. , 2010, Journal of structural biology.

[14]  C. Zimmer,et al.  QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ , 2010, Nature Methods.

[15]  H. Flyvbjerg,et al.  Optimized localization-analysis for single-molecule tracking and super-resolution microscopy , 2010, Nature Methods.

[16]  Abraham J Koster,et al.  Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells. , 2009, European journal of cell biology.

[17]  K. McDonald,et al.  A review of high‐pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues , 2009, Journal of microscopy.

[18]  G. McDermott,et al.  High‐aperture cryogenic light microscopy , 2009, Journal of microscopy.

[19]  Florian Beck,et al.  Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. , 2007, Journal of structural biology.

[20]  J. McIntosh,et al.  Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching , 2007, Journal of microscopy.

[21]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[22]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[23]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[24]  Paul R Selvin,et al.  Polarization effect on position accuracy of fluorophore localization. , 2006, Optics express.

[25]  Michel Orrit,et al.  Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the Ensemble and Single-Molecule Levels , 2004 .

[26]  L E Scriven,et al.  Controlled environment vitrification system: an improved sample preparation technique. , 1988, Journal of electron microscopy technique.

[27]  Songye Chen,et al.  Correlated light and electron cryo-microscopy. , 2010, Methods in enzymology.