Single-molecule localization microscopy analysis with ImageJ

ImageJ is a versatile and powerful tool for quantitative image analysis in microscopy. It is open-source software, platform-independent and enables students and researchers to obtain an easy but thorough introduction into image analysis. Especially the image processing package Fiji is a valuable and powerful extension of ImageJ. Several plugins and macros for single-molecule localization microscopy (SMLM) have been developed during the last decade. These novel tools cover the steps from single-molecule localization and image reconstruction to SMLM data postprocessing such as density analysis, image registration or resolution estimation. This article describes how ImageJ/Fiji can be used for image analysis, reviews existing extensions for SMLM, and aims at introducing and motivating novices and advanced SMLM users alike to explore the possibilities of ImageJ/Fiji for automated and quantitative data analysis

Reply to 'Impact of optical aberrations on axial position determination by photometry'

Franke and van de Linde reply to 'Impact of optical aberrations on axial position determination by photometry'

Parameter-free Molecular Super-Structures Quantification in Single-Molecule Localisation Microscopy

Understanding biological function requires the identification and characterization of complex patterns of molecules. Single-molecule localization microscopy (SMLM) can quantitatively measure molecular components and interactions at resolutions far beyond the diffraction limit, but this information is only useful if these patterns can be quantified and interpreted. We provide a new approach for the analysis of SMLM data that develops the concept of structures and super-structures formed by interconnected elements, such as smaller protein clusters. Using a formal framework and a parameter-free algorithm, (super-)structures formed from smaller components are found to be abundant in classes of nuclear proteins, such as heterogeneous nuclear ribonucleoprotein particles (hnRNPs), but are absent from ceramides located in the plasma membrane. We suggest that mesoscopic structures formed by interconnected protein clusters are common within the nucleus and have an important role in the organization and function of the genome. Our algorithm, SuperStructure, can be used to analyze and explore complex SMLM data and extract functionally relevant information

Light-induced cell damage in live-cell super-resolution microscopy

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities

Single-molecule photoswitching and localization

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution

From single-molecule spectroscopy to super-resolution imaging of the neuron: a review.

For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research.This work was supported by grants from the UK Engineering and Physical Sciences Research Council (EPSRC), The Wellcome Trust, Alzheimer’s Research UK, the Medical Research Council (MRC), and the Biotechnology and Biological Sciences Resesarch Council (BBSRC)

Structural analysis of herpes simplex virus by optical super-resolution imaging.

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.This work was supported by grants from the Leverhulme Trust (grant RPG-2012-793), the Royal Society (University Research Fellowship to C.M.C.), the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1) and by the Medical Research Council (grant MR/K015850/1).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150122/ncomms6980/full/ncomms6980.html

Investigating Cellular Structures at the Nanoscale with Organic Fluorophores

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging

Optimizing photoswitching performance of organic dyes for SMLM through a single MEMS mirror

Whilst SMLM is able to localize molecules with nanometre precision it is only able to achieve this if the imaging parameters have been properly optimised. Key parameters we have investigated for optimisation are homogeneous excitation illumination and the optimal pH and thiol concentrations for photoswitching buffers. Typical SMLM experiments make use of conventional gaussian illumination modes meaning either a compromise in the excitation intensity is made due to overfilling of the objective lens, or an uneven illumination field of view (FOV) is observed which can cause intensity driven photoswitching differences in dye molecules located at different points in the FOV. We demonstrate the use of a single microelectromechanical system (MEMS) mirror as a cost-effective method to generate a flat-field of illumination across the FOV resulting in consistent SMLM metrics. We also show a workflow employing an intensity gradient through the MEMS in which we screen for optimal pH and thiol concentrations to obtain the best results for brightness and photoswitching performances of the carbocyanine dye Alexa Fluor 647. Finally, we have monitored the performance of the oxygen scavenger system based on glucose and glucose oxidase in open or closed environments, determining the amount of acidification present in prolonged imaging experiments