Super-resolution microscopy: going live and going fast

Super-resolution microscopy is increasingly becoming an important tool for biological research, providing valuable information at the nanometer length scales inside cells and tissues. In the past decade numerous technological advancements have transformed superresolution microscopes into powerful tools of discovery. While the first super-resolution images took several hours to acquire, recent progress has led to tremendous improvement in acquisition speed, enabling researchers to probe dynamic processes in living cells with unprecedented spatiotemporal resolution. This mini-review focuses on the recent developments in live-cell super-resolution microscopy and its biological applications.This work was supported in part by Fundació Cellex, Barcelona, Marie-Curie International Reintegration Grant FP7-PEOPLE-2010-RG and the Plan Nacional Grant from the Spanish Ministry (Ministerio de Economia y Competitividad) FIS2012-37753.Peer ReviewedPostprint (author's final draft

Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore

Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.Peer ReviewedPostprint (published version

Quantitative super-resolution microscopy: pitfalls and strategies for image analysis

Super-resolution microscopy is an enabling technology that allows biologists to visualize cellular structures at nanometer length scales using far-field optics. To break the diffraction barrier, it is necessary to leverage the distinct molecular states of fluorescent probes. At the same time, the existence of these different molecular states and the photophysical properties of the fluorescent probes can complicate data quantification and interpretation. Here, we review the pitfalls in super-resolution data analysis that must be avoided for proper interpretation of images.Peer ReviewedPostprint (author's final draft

Super-resolution imaging with stochastic single molecule localization: concepts, technical developments, and biological applications

Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. Microsc. Res. Tech. 77:502–509, 2014. © 2014 Wiley Periodicals, Inc.Peer ReviewedPostprint (author's final draft

Quantifying Protein Copy Number in Super-Resolution Using an Imaging Invariant Calibration

The use of super-resolution microscopy in recent years has revealed that proteins often form small assemblies inside cells and are organized in nanoclusters. However, determining the copy number of proteins within these nanoclusters constitutes a major challenge because of unknown labeling stoichiometries and complex fluorophore photophysics. We previously developed a DNA-origami-based calibration approach to extract protein copy number from super-resolution images. However, the applicability of this approach is limited by the fact that the calibration is dependent on the specific labeling and imaging conditions used in each experiment. Hence, the calibration must be repeated for each experimental condition, which is a formidable task. Here, using cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample, we demonstrate that the DNA-origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any green-fluorescent-protein-fused protein of interest. To do so, we first quantified the copy number of dynein motors within nanoclusters in the cytosol and along the microtubules. Interestingly, this quantification showed that dynein motors form assemblies consisting of more than one motor, especially along microtubules. This quantification enabled us to use the HeLa IC74 cells as a reference sample to calibrate and quantify protein copy number independently of labeling and imaging conditions, dramatically improving the versatility and applicability of our approach

Distance dependent charge separation and recombination in semiconductor/molecular catalyst systems for water splitting

The photoinduced reduction of three Co electrocatalysts immobilised on TiO(2) is 10(4) times faster than the reverse charge recombination. Both processes show an exponential dependence on the distance between the semiconductor and the catalytic core

The stochastic entry of enveloped viruses: Fusion vs. endocytosis

Viral infection requires the binding of receptors on the target cell membrane to glycoproteins, or ``spikes,'' on the viral membrane. The initial entry is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.Comment: 7 pages, 6 figure

Stage III Xanthogranulomatous Pyelonephritis treated with antibiotherapy and percutaneous drainage

Xanthogranulomatous pyelonephritis (XPN) is a rare inflammatory condition usually secondary to chronic obstruction caused by nephrolithiasis and resulting in infection and irreversible destruction of the renal parenchyma. Its standard therapy consists of total or partial nephrectomy. A case of stage III xanthogranulomatous pyelonephritis treated with antibiotherapy and percutaneous drainage is presented in this paper

A DNA Origami Platform for Quantifying Protein Copy Number in Super-Resolution

Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy