Abstract
Single molecule localization microscopy (SMLM) methods produce data in the form of a spatial point pattern (SPP) of all localized emitters. Whilst numerous tools exist to quantify molecular clustering in SPP data, the analysis of fibrous structures has remained understudied. Taking the SMLM localization coordinates as input, we present an algorithm capable of tracing fibrous structures in data generated by SMLM. Based upon a density parameter tracing routine, the algorithm outputs several fibre descriptors, such as number of fibres, length of fibres, area of enclosed regions and locations and angles of fibre branch points. The method is validated in a variety of simulated conditions and experimental data acquired using the image reconstruction by integrating exchangeable single-molecule localization (IRIS) technique. For this, the nanoscale architecture of F-actin at the T cell immunological synapse in both untreated and pharmacologically treated cells, designed to perturb actin structure, was analysed.
Highlights
Conventional fluorescence microscopy methods produce pixelated images representative of the local distribution of chromophores within the sample, convolved with the microscope point spread function (PSF)
The algorithm is based upon a fibre tracing routine that seeks to minimise its weighted density parameter gradient as localizations are assigned to a fibre
Upon encountering antigen presenting cells (APCs) displaying antigenic peptides on surface Major Histocompatibility Complex (MHC), CD4+ Helper T cells form a junction known as the immunological synapse[32]
Summary
Conventional fluorescence microscopy methods produce pixelated images representative of the local distribution of chromophores within the sample, convolved with the microscope point spread function (PSF) This diffraction pattern limits the systems resolving power to ~200 nm, thereby rendering its use limited for the study of cellular ultrastructure on the nanoscale. Algorithm traces a path that minimises this density parameter gradient, constrained by radial and angular restrictions, to create a landscape of fibrous structures This fibre landscape can be interrogated to determine fibre characteristics such as number of fibres, fibre lengths and enclosed meshwork areas. There are many examples in which the characterisation of nanoscale fibrous structures is important, and, imaging of the actin and tubulin cytoskeletons is frequently used to demonstrate super-resolution microscopy systems. We characterise the fibrous mesh properties at the synapse periphery and synapse centre and show that the actin disrupting agent Cytochalasin D results in fewer, shorter fibres and fewer enclosed regions
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