Bernd Rieger

Measuring image resolution in optical nanoscopy

Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the samples spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.

Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors

ErbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after binding of the epidermal growth factor (EGF) and activation of the receptor tyrosine kinase. Specific inhibitors of the erbB1 receptor tyrosine kinase as well as cytochalasin D, a disruptor of the actin cytoskeleton, abolish transport but not free diffusion of the receptor–ligand complex. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking receptors labeled with EGF conjugated to fluorescent quantum dots. Retrograde transport precedes receptor endocytosis, which occurs at the base of the filopodia. Initiation of transport requires the interaction and concerted activation of at least two liganded receptors and proceeds at a constant rate mediated by association with actin. These findings suggest a mechanism by which filopodia detect the presence and concentration of effector molecules far from the cell body and mediate cellular responses via directed transport of activated receptors.

Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution

One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

Accuracy of the Gaussian Point Spread Function model in 2D localization microscopy

The gaussian function is simple and easy to implement as Point Spread Function (PSF) model for fitting the position of fluorescent emitters in localization microscopy. Despite its attractiveness the appropriateness of the gaussian is questionable as it is not based on the laws of optics. Here we study the effect of emission dipole orientation in conjunction with optical aberrations on the localization accuracy of position estimators based on a gaussian model PSF. Simulated image spots, calculated with all effects of high numerical aperture, interfaces between media, polarization, dipole orientation and aberrations taken into account, were fitted with a gaussian PSF based Maximum Likelihood Estimator. For freely rotating dipole emitters it is found that the gaussian works fine. The same, theoretically optimum, localization accuracy is found as if the true PSF were a gaussian, even for aberrations within the usual tolerance limit of high-end optical imaging systems such as microscopes (Marechals diffraction limit). For emitters with a fixed dipole orientation this is not the case. Localization errors are found that reach up to 40 nm for typical system parameters and aberration levels at the diffraction limit. These are systematic errors that are independent of the total photon count in the image. The gaussian function is therefore inappropriate, and more sophisticated PSF models are a practical necessity.

The persistence length of double stranded DNA determined using dark field tethered particle motion

The wormlike chain model describes the micromechanics of semiflexible polymers by introducing the persistence length. We propose a method of measuring the persistence length of DNA in a controllable near-native environment. Using a dark field microscope, the projected positions of a gold nanoparticle undergoing constrained Brownian motion are captured. The nanoparticle is tethered to a substrate using a single double stranded DNA (dsDNA) molecule and immersed in buffer. No force is exerted on the DNA. We carried out Monte Carlo simulations of the experiment, which give insight into the micromechanics of the DNA and can be used to interpret the motion of the nanoparticle. Our simulations and experiments demonstrate that, unlike other similar experiments, the use of nanometer instead of micrometer sized particles causes particle-substrate and particle-DNA interactions to be of negligible effect on the position distribution of the particle. We also show that the persistence length of the tethering DNA can be estimated with a statistical error of 2 nm, by comparing the statistics of the projected position distribution of the nanoparticle to the Monte Carlo simulations. The persistence lengths of 45 single molecules of four different lengths of dsDNA were measured under the same environmental conditions at high salt concentration. The persistence lengths we found had a mean value of 35 nm (standard error of 2.8 nm), which compares well to previously found values using similar salt concentrations. Our method can be used to directly study the effect of the environmental conditions (e.g., buffer and temperature) on the persistence length.

ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation

Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatio-temporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-Δ4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-Δ4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD‐paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single‐photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two‐photon fs pulses in the range of 780–840 nm. Viability in whole‐mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two‐photon activation of paGFP and the use of a paGFP‐fusion protein in investigations of whole organisms.

The role of photon statistics in fluorescence anisotropy imaging

Anisotropy imaging can be used to image resonance energy transfer between pairs of identical fluorophores and, thus, constitutes a powerful tool for monitoring protein homo-association in living single cells. The requirement for only a single fluorophore significantly simplifies biological preparation and interpretation. We use quantitative methods for the acquisition and image processing of anisotropy data that return the expected error of the anisotropy per pixel based on photon statistics. The analysis methods include calibration procedures and allow for a balance in spatial, anisotropy, and temporal resolution. They are featured here with anisotropy images of fluorescent calibration beads and enhanced green fluorescent protein complexes in live cells.

Quantitative Assessment of Gas Cell Development During the Proofing of Dough by Magnetic Resonance Imaging and Image Analysis

Cereal Chem. 80(4):390–395 The structure of bread crumb is an important factor in consumer acceptance of bakery products. The noninvasive monitoring of the gas cell formation during the proofing of dough can aid in understanding the mechanisms governing the crumb appearance in the baked product. The development of gas cells during the proofing of dough was monitored in a noninvasive manner using magnetic resonance imaging (MRI) at 4.7-T. The acquired MRI time series were analyzed quantitatively using image analysis (IA) techniques. The effects of both kneading temperature and mechanical damage by molding were studied. When additional rheological stress was introduced during molding, a more heterogeneous (coarse) gas cell size distribution was observed, and the dough had a smaller specific volume (as measured by MRI). These characteristics were preserved in the bread crumb structure after baking. The fastdeformation during molding also resulted in an isotropic growth of the dough during proofing, whereas slow-deformation during molding resulted in anisotropic growth. This can be related to a better conservation of stress in the dough under a moderate molding operation. A higher temperature during kneading also resulted in a coarser distribution of the gas cells and a smaller MRI specific dough volume. No effect of kneading temperature on the growth anisotropy could be detected, however. This indicates that temperature has a smaller effect on the conservation of stress in the dough than molding. The current work illustrates the capability of MRI/IA for understanding and predicting the influence of food processing parameters on consumer-relevant features in a food product (bread).

The lateral and axial localization uncertainty in super-resolution light microscopy.

A study of the uncertainty of localizing single-molecule emitters for super-resolution light microscopy is presented. Maximum likelihood estimation (MLE) is found to be superior to least-squares fitting for low background levels, but the performance difference between the two methods decreases to a few percent for practical background levels. It is shown that the performance limit of MLE, the Cramér-Rao lower bound, is well described by a concise analytical formula with only spot width and signal and background photon count as input parameters. These predictions for the lateral localization uncertainty are compared with the localization error obtained from repeated localizations of the same single-molecule emitter. Agreement within a few percent is found, thus verifying the validity of the fitting model and the concise analytical approximation. The analysis is extended by novel analytical results for the dependence of the axial localization uncertainty on background level for the astigmatic, bifocal, and double-helix methods.