Holographic Microscopy With Python and HoloPy

Barkley, Solomon; Dimiduk, Thomas G.; Fung, Jerome; Kaz, David M.; Manoharan, Vinothan N.; McGorty, Ryan; Perry, Rebecca W.; Wang, Anna

doi:10.1109/mcse.2019.2923974

Cited by 42 publications

(35 citation statements)

References 17 publications

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“…Thus, we applied the Wang et al model [ 40 ], which was developed to calculate the scattering cross-section of concentric vesicles with an arbitrary size, lipid concentration, membrane thickness, or number of layers. This model uses the open-source light-scattering package HoloPy [ 82 , 83 , 84 , 85 ] (holopy.readthedocs.io/) and is available on GitHub with an illustrative example ( ). In our analyses, we first calculated the EV turbidity spectra corresponding to F1 wells from the absorbance measured in the visible range (400–600 nm) with a 5-nm step using the following Equation (1) [ 40 ]:

where Absorbance (400–600) corresponds to the spectra measured by the plate reader, and BG is the background absorbance, mainly resulting from the plastic interference of the 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

Kaddour

Lyu

Shouman

et al. 2020

IJMS

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Acellular particles (extracellular vesicles and membraneless condensates) have important research, drug discovery, and therapeutic implications. However, their isolation and retrieval have faced enormous challenges, impeding their use. Here, a novel size-guided particle purification liquid chromatography (PPLC) is integrated into a turbidimetry-enabled system for dye-free isolation, online characterization, and retrieval of intact acellular particles from biofluids. The chromatographic separation of particles from different biofluids—semen, blood, urine, milk, and cell culture supernatants—is achieved using a first-in-class gradient size exclusion column (gSEC). Purified particles are collected using a fraction collector. Online UV–Vis monitoring reveals biofluid-dependent particle spectral differences, with semen being the most complex. Turbidimetry provides the accurate physical characterization of seminal particle (Sp) lipid contents, sizes, and concentrations, validated by a nanoparticle tracking analysis, transmission electron microscopy, and naphthopyrene assay. Furthermore, different fractions of purified Sps contain distinct DNA, RNA species, and protein compositions. The integration of Sp physical and compositional properties identifies two archetypal membrane-encased seminal extracellular vesicles (SEV)—notably SEV large (SEVL), SEV small (SEVS), and a novel non-archetypal-membraneless Sps, herein named membraneless condensates (MCs). This study demonstrates a comprehensive yet affordable platform for isolating, collecting, and analyzing acellular particles to facilitate extracellular particle research and applications in drug delivery and therapeutics. Ongoing efforts focus on increased resolution by tailoring bead/column chemistry for each biofluid type.

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where Absorbance (400–600) corresponds to the spectra measured by the plate reader, and BG is the background absorbance, mainly resulting from the plastic interference of the 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

Kaddour

Lyu

Shouman

et al. 2020

IJMS

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“…This code calculates the exact scattered field (up to series truncation) from a cluster of arbitrary, non-overlapping spheres as a series expansion in vector spherical harmonics. The SCSMFO1B code is wrapped into the open-source package HoloPy (version 3.2.1), which allows for convenient generation and analysis of holograms [24].…”

Section: Calculation Of Exact Hologramsmentioning

confidence: 99%

Computational assessment of an effective-sphere model for characterizing colloidal fractal aggregates with holographic microscopy

Fung

Hoang

2019

Journal of Quantitative Spectroscopy and Radiative Transfer

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We perform simulations to evaluate a recent experimental technique for using in-line holographic microscopy and an effective-sphere model to measure the population-averaged fractal dimension D f of an ensemble of colloidal fractal aggregates. In this technique, models based on Lorenz-Mie scattering by a uniform sphere are fit to digital holograms of a population of fractal aggregates to determine the effective refractive indices n ef f and effective radii a ef f of the aggregates. A scaling relationship between n ef f and a ef f based on the Maxwell Garnett effective-medium theory then determines D f . Here we use a multisphere superposition code to calculate the exact holograms produced by aggregates with tunable fractal dimensions D f . We show that n ef f and a ef f become less sensitive to the aggregate orientation as D f increases. We also show that the Maxwell Garnett scaling relationship correctly determines D f to within 10.5% when multiple scattering is negligible and the population-averaged coefficient of determination R 2 p > 0.6, indicating that the holograms are well-described by the effective-sphere model.

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“…Here, we circumvent these limitations by using a numerically stable multilayered-sphere light-scattering solution (25,26) that explicitly specifies the bilayers and interbilayer spaces to calculate how vesicles scatter light. Because the exact Lorenz-Mie solution for spheres was extended to core-shell spheres more than half a century ago (27), there have been many improvements on algorithms and computational power (28) to make the solution usable.…”

Section: Introductionmentioning

confidence: 99%

Core-Shell Modeling of Light Scattering by Vesicles: Effect of Size, Contents, and Lamellarity

Wang

Miller

Szostak

2019

Biophysical Journal

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Having a fast, reliable method for characterizing vesicles is vital for their use as model cell membranes in biophysics, synthetic biology, and origins of life studies. Instead of the traditionally used Rayleigh-Gans-Debye approximation, we use an exact extended Lorenz-Mie solution for how core-shell particles scatter light to model vesicle turbidity. This approach enables accurate interpretations of simple turbidimetric measurements and is able to accurately model highly scattering vesicles, such as larger vesicles, those with multiple layers, and those with encapsulated material. We uncover several surprising features, including that vesicle lamellarity has a larger effect on sample turbidity than vesicle size and that the technique can be used to measure the membrane thickness of vesicles. We also examine potential misinterpretations of turbidimetry and discuss when measurements are limited by forward and multiple scattering and by the geometry of the instrument.

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Holographic Microscopy With Python and HoloPy

Cited by 42 publications

References 17 publications

Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

Computational assessment of an effective-sphere model for characterizing colloidal fractal aggregates with holographic microscopy

Core-Shell Modeling of Light Scattering by Vesicles: Effect of Size, Contents, and Lamellarity

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