Accurate localization microscopy by intrinsic aberration calibration

Copeland, Craig R.; McGray, Craig D.; Ilić, B.; Geist, Jon; Stavis, Samuel M.

doi:10.1038/s41467-021-23419-y

Cited by 10 publications

(7 citation statements)

References 53 publications

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“…Neither aspect exactly matches the experimental scheme of diffusion in a confining cylinder. As well, optical microscopy throughout a wide 16 and deep 17 focal volume requires calibration of scale and distortion artifacts to correct apparent trajectories that are the source data for 𝐷𝐷 || . Finally, a formal measurement requires accurate estimates of not only input quantities but also input uncertainties, and their propagation through the measurement function to an output quantity and output uncertainty, 18 which is incomplete in Reference 1.…”

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confidence: 99%

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles

et al. 2021

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Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

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confidence: 99%

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles

et al. 2021

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“…Beyond the edge threshold, calibrations of magnification and distortion are necessary for accurate localization (Figure A) . Imaging near best focus and accounting for field dependences are important for such calibrations. ,, We align our reference objects, optimize the imaging conditions for each object (Supporting Information), and image reference objects and microdroplets through focus in axial increments of 1 μm. Collection of each series of images through focus occurs in the same direction of travel and with 100 replicate images at each z position.…”

Section: Resultsmentioning

confidence: 99%

Sub-picoliter Traceability of Microdroplet Gravimetry and Microscopy

et al. 2021

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Gravimetry typically lacks the resolution to measure single microdroplets, whereas microscopy is often inaccurate beyond the resolution limit. To address these issues, we advance and integrate these complementary methods, introducing simultaneous measurements of the same microdroplets, comprehensive calibrations that are independently traceable to the International System of Units (SI), and Monte-Carlo evaluations of volumetric uncertainty. We achieve sub-picoliter agreement of measurements of microdroplets in flight with volumes of approximately 70 pL, with ensemble gravimetry and optical microscopy both yielding 95% coverage intervals of ±0.6 pL, or relative uncertainties of ±0.9%, and root-mean-square deviations of mean values between the two methods of 0.2 pL or 0.3%. These uncertainties match previous gravimetry results and improve upon previous microscopy results by an order of magnitude. Gravimetry precision depends on the continuity of droplet formation, whereas microscopy accuracy requires that optical diffraction from an edge reference matches that from a microdroplet. Applying our microscopy method, we jet and image water microdroplets suspending fluorescent nanoplastics, count nanoplastic particles after deposition and evaporation, and transfer volumetric traceability to the number concentrations of single microdroplets. We expect that our methods will impact diverse fields involving dimensional metrology and volumetric analysis of microdroplets, including inkjet microfabrication, disease transmission, and industrial sprays.

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“…In the subsequent applications of the measurement method in ref , a different objective lens and imaging conditions prolong the tracking analysis from 600 to 2000 images. Although longer trajectories could reduce the error of the standard deviation by up to a factor of , different systematic effects could also be present due to scale factor and aberration artifacts that are intrinsic to objective lenses. , As well, ref notes potential errors due to a hard-sphere analysis of soft-lipid nanoparticles with complex diffusive behavior. This mismatch of experimental conditions and potential errors disconnects the efforts to validate and apply the measurement method.…”

Section: Measurement Functionmentioning

confidence: 99%

“…Regarding C/D || , the hydrodynamic correction 12,13 applies only at the midplane of a slit and deviates significantly from the results of careful measurements of microparticle diffusion near the surface of a slit, 14 representative model fit systematically underestimates the experimental data at short lag times (Figure 1D of ref 1). As well, optical microscopy throughout a wide 16 and deep 17 focal volume requires calibration of scale factor and aberration artifacts such as distortion and defocus to correct apparent trajectories that are the source data for D || . Finally, a formal measurement requires accurate estimates of not only input quantities but also input uncertainties, and their propagation through the measurement function to an output quantity and output uncertainty, 18 which is incomplete in ref 1.…”

mentioning

confidence: 99%