Subnanometer localization accuracy in widefield optical microscopy
📝 Abstract
The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue - overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.
💡 Analysis
The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue - overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.
📄 Content
1
Subnanometer localization accuracy in widefield optical microscopy
Craig R. Copeland1, 2, Jon Geist3, Craig D. McGray3, Vladimir A. Aksyuk1, J. Alexander Liddle1, B. Robert Ilic1, and Samuel M. Stavis1, *
1Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States of America, 2Maryland NanoCenter, University of Maryland, College Park, Maryland 20742, United States of America, 3Engineering Physics Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States of America
Abstract: The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue – overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.
Keywords: critical dimension; localization accuracy; nanoparticle fiducial; optical microscopy; reference material
INTRODUCTION Optical microscopy methods of localizing small emitters are broadly useful in such fields as cell biology, nanoscale fabrication, cryogenic physics, and microelectromechanical systems1. Both precision2, 3, 4 and accuracy are fundamental to localization microscopy5, 6. Localization of single fluorophores with a statistical uncertainty of tens of nanometers is common, and subnanometer uncertainty is possible for fluorophores7 and readily achievable for brighter emitters such as particles8. Whereas improving localization precision generally requires counting more signal photons by increasing the intensity and stability of emission9, 10, achieving commensurate localization accuracy presents diverse challenges in the calibration of an optical microscope as a nonideal measurement system. Such calibration involves not only the discrete parts of the system but also the interaction of those parts during a measurement and is rarely, if ever, implemented. This can cause overconfidence in measurement results with statistical uncertainties at the nanometer scale that are invalid due to larger systematic errors. These errors can extend into the micrometer scale when localizing emitters across a wide field, as is often necessary for imaging microstructures and tracking motion11, 12. The discrepancy between precision and accuracy can be so large as to require a logarithmic target to illustrate, as Figure 1 shows.
2
Figure 1. (a) Schematic showing a linear target. (b) Schematic showing a logarithmic target. Green dots are localization data. Their scatter indicates statistical uncertainty at the subnanometer scale, which is not apparent on the linear target as systematic errors can be four orders of magnitude larger. This discrepancy requires a logarithmic target to illustrate both precision and accuracy. Calibration of the measurement system and correction of localization data ensures that precision is the limit of accuracy13.
The root cause of the problem is a lack of reference materials and calibration methods that are optimal for localization microscopy, analogous to those for optical imaging at larger scales14. Small particles are useful for mapping certain effects of optical aberrations15, 16, 17. However, their size distribution and random deposition can result in nonuniform sampling of the imaging field, fluorophores in particles often have a different emission spectrum from that of fluorophores in solution, and evaluating magnification18 requires a specification of distance between emitters. DNA origami can control the submicrometer distance between a few fluorophores19, 20, but this approach has limitations of emitter intensity and stability, as well
This content is AI-processed based on ArXiv data.