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.
There is considerable interest in using Quantum Dots (QDs) as fluorescent probes such for cellular imaging due to unique advantages in comparison with conventional molecular dyes. However, cytosolic delivery of QDs into live cells remains a major challenge. Here we demonstrate highly efficient delivery of PEG-coated QDs into live cells by means of laser-induced vapour nanobubbles. Using this procedure we succeeded in high-throughput loading of ~80% of cells while maintaining a cell viability of ~85%.
Macromolecular agents such as nucleic acids and proteins need to be delivered into living cells for therapeutic purposes. Among physical methods to deliver macromolecules across the cell membrane, laser-induced photoporation using plasmonic nanoparticles is a method that is receiving increasing attention in recent years. By irradiating gold nanoparticles bound to the cell membrane with laser light, nanosized membrane pores can be created. Pores are formed by localized heating or by vapour nanobubbles (VNBs) depending on the incident laser energy. Macromolecules in the surrounding cell medium can then diffuse through the transiently formed pores into the cytoplasm. While both heating and VNBs have been reported before for permeabilization of the cell membrane, it remains unclear which of both methods is more efficient in terms of cell loading with minimal cytotoxicity. In this study we report that under condition of a single 7 ns laser pulse VNBs are substantially more efficient for the cytosolic delivery of macromolecules. We conclude that VNB formation is an interesting photoporation mechanism for fast and efficient macromolecular delivery in live cells.
KEYWORDS: Diffusion, Luminescence, Point spread functions, Particles, Data modeling, Proteins, Monte Carlo methods, Image resolution, Biomedical optics, Medical research
Fluorescence recovery after photobleaching (FRAP) is a common technique to probe mobility of fluorescently labeled proteins in biological membranes by monitoring the time-dependence of the spatially integrated fluorescence signals after a bleaching pulse. Discrimination by FRAP between free diffusion with an immobile fraction (FDIM) and the phenomenological model for anomalous diffusion based on the time-dependent diffusion coefficient (TDDC) is a challenging problem, requiring extremely long observation times for differentiation. Recently, rectangular FRAP (rFRAP) has been introduced for normal diffusion by considering not only the temporal but also spatial information, taking the effective point spread function of the optical system into account. In this work we provide an extension of rFRAP toward anomalous diffusion according to the continuous time random walk (CTRW). We explore whether the spatial information in rFRAP allows for enhanced discrimination between FDIM, TDDC, and CTRW in a single experiment within a feasible time window. Simulations indicate that rFRAP can indeed differentiate the different models by evaluating the spatial autocorrelation of the differences between the measured and fitted pixel values. Hence, rFRAP offers a tool that is capable of discriminating different types of diffusion at shorter time scales than in the case where spatial information is discarded.
The present work describes the use of a recently established multiparametric methodology to study nanomaterial
toxicity. Using optimized methods, including proliferation-restricted cell types and endosomal buffer systems, the
effect of different types of nanomaterials on cultured cells were studied, focusing in particular on intracellular
particle degradation. Gold particles were quite resistant, whereas iron oxide degraded, with loss of magnetic
resonance contrast, but little toxicity associated. Quantum dots degraded more slowly, decreasing both fluorescence
quantum yield and cell viability over long-time periods. The multiparametric methodology is shown to be an
efficient screening strategy, allowing easy comparison of results obtained for different nanomaterials and hereby
helping to optimize nanoparticle design with improved safety.
Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients.
Kevin Braeckmans, Kevin Buyens, Wim Bouquet-Geerardyn, Chris Vervaet, Philippe Joye, Filip De Vos, Laurent Plawinski, Loïc Doeuvre, Eduardo Angles-Cano, Niek Sanders, Jo Demeester, Stefaan De Smedt
While extremely relevant to many life science fields, such as biomedical diagnostics and drug delivery, studies on the
size of nanoparticulate matter dispersed in biofluids are missing due to a lack of suitable methods. Here we report that
fluorescence single particle tracking (fSPT) with maximum entropy analysis is the first technique suited for accurate
sizing of nanoparticles dispersed in biofluids, such as whole blood. After a thorough validation, the fSPT sizing method
was applied to liposomes that have been under investigation for decades as nanocarriers for drugs. The tendency of these
liposomes to form aggregates in whole blood was tested in vitro and in vivo. In addition, we have demonstrated that the
fSPT sizing technique can be used for identifying and sizing natural cell-derived microparticles directly in plasma. fSPT
sizing opens up the possibility to systematically study the size and aggregation of endogenous or exogenous
nanoparticles in biofluids.
In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.
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