Advancing evanescent light scattering microscopy for single-particle characterization of gene delivery nanoparticles

Published

March 4, 2026

Author(s)

Jagat Budhathoki, Gregory Cooksey, Matthew DiSalvo, Thomas Germer, Peter Bajcsy, Edward Kwee, Aaron Goldfain, Alexander Peterson

Abstract

Precise and reproducible single-nanoparticle characterization is essential for advancing gene therapy, where viral vectors encapsulating therapeutic genes must be evaluated under formulation-relevant conditions. We present a label-free optical platform based on dark-field total internal reflection microscopy (DF-TIRM), developed to image and quantitatively characterize nanoparticles near a passivated glass–water interface using evanescent illumination. The system employs a prism-based total internal reflection (TIR) geometry to selectively illuminate near-surface particles, enabling high-contrast scattering detection while rejecting incident light. Using polystyrene nanospheres with a diameter of 100 nm as a well-defined model system, we systematically investigate how solution conditions and glass surface functionalization influence undesirable nonspecific particle sticking over a range of NaCl concentrations. On bare glass, polystyrene beads can be imaged without significant sticking only at very low ionic strength (≤ 1 mmol/L NaCl); above 10 mmol/L, immobilized particles rapidly accumulate and overcrowd the surface. Supported lipid bilayers markedly reduce sticking at intermediate salt (20 mmol/L to 50 mmol/L) but still accumulate immobilized beads at 100–150 mmol/L. Polyethylene glycol (PEG) treated (PEGylated) glass, in our implementation, performs poorly without surfactant, exhibiting even higher sticking than bare glass at 20 mmol/L. Finally, we show that adding a volume fraction of 0.1% Polysorbate 20 to 150 mmol/L NaCl dramatically reduces sticking on both bilayer and PEG-coated surfaces. These results provide practical guidelines for selecting surface and buffer conditions that preserve suspended particles within the evanescent field and lay the groundwork for future DF-TIRM measurements on viral gene-delivery vectors.

Proceedings Title

Advancing evanescent light scattering microscopy for single-particle characterization of gene delivery nanoparticles

Volume

13852

Conference Dates

January 17-22, 2026

Conference Location

San Franscisco, CA, US

Conference Title

SPIE Photonic West

Pub Type

Conferences

Download Paper

https://doi.org/10.1117/12.3080845

Local Download

Keywords

Evanescent light-scattering microscopy, dark-field total internal reflection microscopy, gene delivery nanoparticles, viral vectors, surface passivation, supported lipid bilayers, PEGylated glass, Polysorbate 20

Citation

Budhathoki, J. , Cooksey, G. , DiSalvo, M. , Germer, T. , Bajcsy, P. , Kwee, E. , Goldfain, A. and Peterson, A. (2026), Advancing evanescent light scattering microscopy for single-particle characterization of gene delivery nanoparticles, Advancing evanescent light scattering microscopy for single-particle characterization of gene delivery nanoparticles, San Franscisco, CA, US, [online], https://doi.org/10.1117/12.3080845, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=961236 (Accessed March 24, 2026)