Quantitative evaluation of software packages for single-molecule localization microscopy

The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs.

Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

Paolo Annibale

When fluorophores are densely packed in a biological sample, localizing them one at a time is one of the paths to optical super-resolution. Photo Activated Localization Microscopy (PALM) is one of the methods of this burgeoning field that presents a large promise for addressing physical and biological questions requiring non-invasive, high-resolution optical imaging at the nanoscale. Since this work began when PALM was only two years old, the thesis contains a significant portion of research on the technique itself, and has therefore a marked methodological cut. The principal, and recurring, theme is the investigation of the single molecule fluorescence dynamics of the fluorophores used, that in the end determine to a large extent the ensemble imaging performance as well as the interpretation of the data. A few assumptions reported in the literature are questioned, and, beginning with a discussion of the properties of a bright fluorescent protein, mEos2, we propose an original approach based on the use of the temporal information besides the spatial one when treating PALM data. This allows both a more accurate quantification of the number of fluorophores activated in the sample as well as the correct identification of relevant biological structures, such as clusters, on the plasma membrane of cells. The second theme is the application of PALM to probe the functional arrangement of an important class of cell membrane proteins, through the study of the prototypical G protein-coupled receptor [beta]2-Adrenergic Receptor. First, the well studied biological properties of this family of signaling proteins are used to validate the potential of this approach. Then, PALM is used to gain new insight on the basal arrangement of [beta]2-AR in a relevant biological context using tools from spatial point pattern analysis. Provided that appropriate control experiments are performed, PALM is shown to have the potential to be included in the palette of the available techniques to study membrane protein organization. An important finding is the quantification of a cell-type specific clustering of this receptor in cardiomyocite-like cells. Finally, some technical issues relevant to dual color imaging, in particular concerning the axial stability of the microscope, are addressed as a third, perspective theme, together with a detailed first-time characterization of three representative fluorescent protein pairs currently available for dual color PALM imaging.

EPFL2012

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

Anurag Agrawal, Anna Archetti, Silvia Colabrese, Ricardo Henriques, Seamus John Holden, Tomas Lukes, Thomas Pengo, Thanh-An Michel Pham, Daniel Sage, Michaël Unser

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.

2019

Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

Paolo Annibale

EPFL2012