Nanoparticle-based applications require increasingly sensitive screening tools, necessitating single-particle, multi-parameter analysis techniques in the native liquid environment. In this work, we present a laser-scanning camera using acousto-optic deflectors and a single-photon counting module. By scanning a highly focused laser beam (532 nm) across a grid of pixels, and by detecting the associated emitted photons with high sensitivity, fluorescent nanoparticles of 100 nm and below can be imaged at frame rates up to 12 kHz.
The development of nanoparticle-based applications relies increasingly on our ability to characterize nanoparticles at the single-particle level and in their native liquid environment. In quantum dot applications, for example, a detailed understanding of how spectral properties are related to differences in size, shape, composition and electrical charge is crucial to optimize the fabrication process of quantum dots and ultimately their photonic or biomedical applications. Since deposition on a substrate may disturb the spectroscopic response it is preferred that measurements occur in the liquid phase. Sufficiently fast imaging is needed for measurements of the electrical charge and particle size. In the present work, we focus on measuring the electrophoretic mobility and size of individual nanoparticles in their native liquid environment through sensitive imaging at frame rates up to 10 kHz in the presence of an electric field. The particles of interest are fluorescently labelled polystyrene particles in the range of 40 nm to 100 nm. The imaging method is based on scanning a focused laser beam (532 nm) across a 10 µm x 10 µm field using acousto-optic deflectors and by detecting the emitted fluorescent light with a single-photon counter. The particle size is determined from Brownian motion analysis. The electrophoretic mobility and the electroosmotic mobility related to the zeta-potential of the microchannel walls are determined through analysis of the response at different frequencies of the applied electric field and at different positions in the microfluidic channel.
Endosomal escape remains the most prominent bottleneck at the intracellular level for non-viral vectors today. Gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation. Depending on the energy of nanosecond laser pulses, this can be achieved by either endosomal rupture by the mechanical energy from water vapor nanobubbles (VNBs) that emerge around the AuNP, or permeabilization of the endosomal membrane by heat diffusion. Here, we designed a siRNA/AuNP drug delivery system, to address the open question of how both photothermal effects influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. We found that, contrary to heat-mediated endosomal escape, VNB generation produced excellent transfection levels independent of the cell type, without inducing long-term changes in cell homeostasis.
An important reason for the decreased sensitivity of bacteria towards antibiotics is their capability to form so-called biofilms. The increased tolerance of sessile cells is multifactorial and includes reduced penetration rate and potency of antibiotics through dense biofilms. Strategies that have the ability to interfere with the integrity of biofilms and allowing a better penetration of antimicrobial agents are highly sought after. In this work, we explore the potential of laser-induced vapor nanobubbles (VNB) formed from plasmonic nanoparticles irradiated with nanosecond pulsed laser light to locally disturb biofilm integrity and improve antibiotics diffusion. Our results show that biofilms of both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria can be locally disrupted by the pressure waves from laser-induced VNB inside the biofilms. Most importantly, VNB-mediated biofilm disruption increased tobramycin efficacy up to 1-3 log orders of magnitude, depending on the treatment regimens and type of organism. In addition, we explored the use of VNB to enhance the efficacy of a broad range of antimicrobials used for treating wound infections, towards a first potential clinical application of the technique. Our results confirm that VNB-mediated biofilm disruption is an effective technique to enhance the activity of those antimicrobials that experience hindered diffusion in biofilms. Future work will extend the evaluation of this novel concept towards more complex multi-species biofilms and in vitro wound models before going to in vivo evaluations.
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