Mark Schüttpelz

Real-Time Computation of Subdiffraction-Resolution Fluorescence Images

In the recent past, single‐molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction‐resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high‐precision fluorophore localization and subsequent visualization of found fluorophore positions in a super‐resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non‐maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real‐time data processing is possible and that super‐resolution imaging with organic fluorophores of cellular structures with ∼20 nm optical resolution can be completed in less than 10 s.

Reversible Photoswitchable DRONPA‐s Monitors Nucleocytoplasmic Transport of an RNA‐Binding Protein in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non‐fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon‐optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA‐s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA‐s fused to the RNA‐binding protein AtGRP7 (Arabidopsis thaliana glycine‐rich RNA‐binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7‐DRONPA‐s, we set up a single‐molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7‐DRONPA‐s reaches the nucleus by carrier‐mediated transport. Furthermore, photoactivation experiments showed that AtGRP7‐DRONPA‐s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA‐s is a versatile tool to track protein transport dynamics in stably transformed plants.

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.

Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy

Liver sinusoidal endothelial cells (LSEC) are an important class of endothelial cells facilitating the translocation of lipoproteins and small molecules between the liver and blood. A number of clinical conditions, especially metabolic and aging-related disorders, are implicated by improper function of LSECs. Despite their importance, research into these cells is limited because the primary ultrastructures involved in their function are transcellular pores, called fenestrations, with diameters in a size range between 50-200 nm, i.e. well below the optical diffraction limit. Here, we show that we are able to resolve fenestrations with a spatial resolution of ∼20 nm by direct stochastic optical reconstruction microscopy (dSTORM). The cellular plasma membrane was labeled at high fluorophore density with CellMask Deep Red and imaged using a reducing buffer system. We compare the higher degree of structural detail that dSTORM provides to results obtained by 3D structured illumination microscopy (3D-SIM). Our results open up a path to image these physiologically important cells in vitro using highly resolving localization microscopy techniques that could be implemented on non-specialized fluorescence microscopes, enabling their investigation in most biomedical laboratories without the need for electron microscopy.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

Nanoscopy of bacterial cells immobilized by holographic optical tweezers

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscopes image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.

dSTORM: real-time subdiffraction-resolution fluorescence imaging with organic fluorophores

In the recent past, a variety of methods have been developed to circumvent the diffraction barrier of light which restricts optical resolution to about 200 nm in the image plane. Single-molecule based photoswitching microscopy such as direct stochastic optical reconstruction microscopy (dSTORM) has been successfully implemented for subdiffraction-resolution fluorescence imaging. The major drawback of this technique has been that the reconstruction of subdiffraction-resolution images requires substantially more time than the actual experiment and prevented real-time imaging. Here we present a new computational algorithm enabling subdiffraction-resolution fast imaging of cellular structures with ~20 nm optical resolution in less than 10 seconds.

Photoswitching microscopy with subdiffraction-resolution

High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable fluorescent probes. We extended this principle for carbocyanines without the need of an activator fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (dSTORM). Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states. Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565, or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular compartments and can serve as a tool for molecular quantification.

Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy – high performance super-resolution at low cost

Many commercial as well as custom-built fluorescence microscopes use scientific-grade cameras that represent a substantial share of the instrument’s cost. This holds particularly true for super-resolution localization microscopy where high demands are placed especially on the detector with respect to sensitivity, noise, and also image acquisition speed. Here, we present and carefully characterize an industry-grade CMOS camera as a cost-efficient alternative to commonly used scientific cameras. Direct experimental comparison of these two detector types shows widely similar performance for imaging by single molecule localization microscopy (SMLM). Furthermore, high image acquisition speeds are demonstrated for the CMOS detector by ultra-fast SMLM imaging.

A reliable and sensitive bead-based fluorescence assay for identification of nucleic acid sequences

The sensitive and rapid detection of pathogenic DNA is of tremendous importance in the field of diagnostics. We demonstrate the ability of detecting and quantifying single- and double-stranded pathogenic DNA with picomolar sensitivity in a bead-based fluorescence assay. Selecting appropriate capturing and detection sequences enables rapid (2 h) and reliable DNA quantification. We show that synthetic sequences of S. pneumoniae and M. luteus can be quantified in very small sample volumes (20 μL) across a linear detection range over four orders of magnitude from 1 nM to 1 pM, using a miniaturized wide-field fluorescence microscope without amplification steps. The method offers single molecule detection sensitivity without using complex setups and thus volunteers as simple, robust, and reliable method for the sensitive detection of DNA and RNA sequences.