Dataset Information

Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy.

ABSTRACT: Many single nanoemitters such as fluorescent molecules produce dipole radiation that leads to systematic position errors in both particle tracking and super-resolution microscopy. Via vectorial diffraction equations and simulations, we show that imaging only azimuthally polarized light in the microscope naturally avoids emission from the z-component of the transition dipole moment, resulting in negligible localization errors for all emitter orientations and degrees of objective lens misfocus. Furthermore, localization accuracy is maintained even in the presence of aberrations resulting from imaging in mismatched media.

SUBMITTER: Lew MD

PROVIDER: S-EPMC4245985 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy.

Lew Matthew D MD Moerner W E WE

Nano letters 20141016 11

Many single nanoemitters such as fluorescent molecules produce dipole radiation that leads to systematic position errors in both particle tracking and super-resolution microscopy. Via vectorial diffraction equations and simulations, we show that imaging only azimuthally polarized light in the microscope naturally avoids emission from the z-component of the transition dipole moment, resulting in negligible localization errors for all emitter orientations and degrees of objective lens misfocus. Fu ...[more]

PMID: 25272093

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Project description:Single molecule localization microscopy has become a prominent technique to quantitatively study biological processes below the optical diffraction limit. By fitting the intensity profile of single sparsely activated fluorophores, which are often attached to a specific biomolecule within a cell, the locations of all imaged fluorophores are obtained with ∼20 nm resolution in the form of a coordinate table. While rendered super-resolution images reveal structural features of intracellular structures below the optical diffraction limit, the ability to further analyze the molecular coordinates presents opportunities to gain additional quantitative insights into the spatial distribution of a biomolecule of interest. For instance, pair-correlation or radial distribution functions are employed as a measure of clustering, and cross-correlation analysis reveals the colocalization of two biomolecules in two-color SMLM data. Here, we present an efficient filtering method for SMLM data sets based on pair- or cross-correlation to isolate localizations that are clustered or appear in proximity to a second set of localizations in two-color SMLM data. In this way, clustered or colocalized localizations can be separately rendered and analyzed to compare other molecular properties to the remaining localizations, such as their oligomeric state or mobility in live cell experiments. Current matrix-based cross-correlation analyses of large data sets quickly reach the limitations of computer memory due to the space complexity of constructing the distance matrices. Our approach leverages k-dimensional trees to efficiently perform range searches, which dramatically reduces memory needs and the time for the analysis. We demonstrate the versatile applications of this method with simulated data sets as well as examples of two-color SMLM data. The provided MATLAB code and its description can be integrated into existing localization analysis packages and provides a useful resource to analyze SMLM data with new detail.

Project description:Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

Dataset Information

Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy.

Publications

Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy.

Similar Datasets

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