A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H.
Nat Methods. 2012 Jun;9(6):582-4. doi: 10.1038/nmeth.1991. Epub 2012 Apr 29.
We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.
Binding-activated localization microscopy of DNA structures.
Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V.
Nano Lett. 2011 Sep 14;11(9):4008-11. Epub 2011 Aug 18.
Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of approximately 14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.
Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.
Ries, J., Yu, S.R., Burkhardt, M., Brand, M. & Schwille, P.
Nat Methods. 2009 Sep;6(9):643-5. Epub 2009 Aug 2.
Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.
Supercritical angle fluorescence correlation spectroscopy.
Ries, J., Ruckstuhl, T., Verdes, D. & Schwille, P.
Biophys J. 2008 Jan 1;94(1):221-9. Epub 2007 Sep 7.
We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.