Mark Bates

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.

The 2015 super-resolution microscopy roadmap.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to ...

mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001

Super-resolution microscopy of clickable amino acids reveals the effects of fluorescent protein tagging on protein assemblies.

The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging.

A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.

A new approach to fluorescence microscopy.

Precise localization of switchable fluorescent molecules facilitates nanoscale biological imaging. The fluorescence microscope is a powerful tool for the study of molecular and cell biology, enabling researchers to peer into cells and living organisms to understand their spatial organization. Methods for tagging specific biomolecules with fluorescent labels, such as green fluorescent protein (GFP), provide a toolbox for the observation of protein organization, interactions, and dynamics (1). This enables the visualization of a host of biological phenomena, such as enzymatic processes, the development of cells and organisms, and the structural dynamics of individual biomolecules.

Gpufit: An open-source toolkit for GPU-accelerated curve fitting

We present a general purpose, open-source software library for estimation of non-linear parameters by the Levenberg-Marquardt algorithm. The software, Gpufit, runs on a Graphics Processing Unit (GPU) and executes computations in parallel, resulting in a significant gain in performance. We measured a speed increase of up to 42 times when comparing Gpufit with an identical CPU-based algorithm, with no loss of precision or accuracy. Gpufit is designed such that it is easily incorporated into existing applications or adapted for new ones. Multiple software interfaces, including to C, Python, and Matlab, ensure that Gpufit is accessible from most programming environments. The full source code is published as an open source software repository, making its function transparent to the user and facilitating future improvements and extensions. As a demonstration, we used Gpufit to accelerate an existing scientific image analysis package, yielding significantly improved processing times for super-resolution fluorescence microscopy datasets.

Single-particle analysis for fluorescence nanoscopy.

Building on methods from electron microscopy, researchers are applying nanoscale fluorescence imaging to address questions in structural biology.

Q-STORM: Quantitative Super-resolution Microscopy from Localization of Reversibly Switchable Single-Molecules

We make localization microscopy with reversibly switchable fluorophores a quantitative imaging technique by measuring the average number of activation events per marker from the buildup of spatial image correlations during image acquisition.