Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent performance. This approach can easily be integrated with advanced multimodal live-cell microscopy and micromanipulation techniques. The efficiency of this technique depends on an understanding by the user of both biology and physics. Therefore, in this protocol we discuss the subtleties that apply to both fields, including sample preparation, alignment and calibration of laser optics and their integration into a microscopy platform. The entire protocol takes ~5 d to complete, from the initial setup of the femtosecond optical transfection system to the final stage of fluorescence imaging to assay for successful expression of the gene of interest.
We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for the controlled and enhanced optoinjection and phototransfection of mammalian cells with a femtosecond light source. The SLM provides full control over the lateral and axial positioning of the beam with sub‐micron precision. Fast beam translation enables time‐sequenced irradiation, which is shown to enhance the optoinjection efficiency and alleviate the problem of exact beam positioning on the cell membrane. We show that irradiation in three axial positions doubles the number of viably optoinjected cells when compared with a single dose. The presented system also enables untargeted raster scan irradiation which provides a higher throughput transfection of adherent cells at the rate of 1 cell per second. Additionally, fluorescent imaging is used to demonstrate cell selective two‐step gene therapy. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
We demonstrate laser-induced breakdown of an optically trapped nanoparticle with a nanosecond laser pulse. Controllable cavitation within a microscope sample was achieved, generating shear stress to monolayers of live cells. This efficiently permeabilize their plasma membranes. We show that this technique is an excellent tool for plasmid-DNA transfection of cells with both reduced energy requirements and reduced cell lysis compared to previously reported approaches. Simultaneous multisite targeted nanosurgery of cells is also demonstrated using a spatial light modulator for parallelizing the technique.
Hydrogel waveguides have found increased use for variety of applications where biocompatibility and flexibility are important. In this work, we demonstrate the use of polyethylene glycol diacrylate (PEGDA) waveguides to realize a monolithic lab-on-a-chip device. We performed a comprehensive study on the swelling and optical properties for different chain lengths and concentrations in order to realize an integrated biocompatible waveguide in a microfluidic device for chemical sensing. Waveguiding properties of PEGDA hydrogel were used to guide excitation light into a microfluidic channel to measure the fluorescence emission profile of rhodamine 6G as well as collect the fluorescence signal from the same device. Overall, this work shows the potential of hydrogel waveguides to facilitate delivery and collection of optical signals for potential use in wearable and implantable lab-on-a-chip devices.
A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.
BackgroundCell-penetrating peptides (CPPs) can act as carriers for therapeutic molecules such as drugs and genetic constructs for medical applications. The triggered release of the molecule into the cytoplasm can be crucial to its effective delivery. Hence, we implemented and characterized laser interaction with defined gold nanoparticle agglomerates conjugated to CPPs which enables efficient endosomal rupture and intracellular release of molecules transported.ResultsGold nanoparticles generated by pulsed laser ablation in liquid were conjugated with CPPs forming agglomerates and the intracellular release of molecules was triggered via pulsed laser irradiation ( = 532 nm, = 1 ns). The CPPs enhance the uptake of the agglomerates along with the cargo which can be co-incubated with the agglomerates. The interaction of incident laser light with gold nanoparticle agglomerates leads to heat deposition and field enhancement in the vicinity of the particles. This highly precise effect deagglomerates the nanoparticles and disrupts the enclosing endosomal membrane. Transmission electron microscopy images confirmed this rupture for radiant exposures of 25 mJ/cm and above. Successful intracellular release was shown using the fluorescent dye calcein. For a radiant exposure of 35 mJ/cm we found calcein delivery in 81 % of the treated cells while maintaining a high percentage of cell viability. Furthermore, cell proliferation and metabolic activity were not reduced 72 h after the treatment.ConclusionCPPs trigger the uptake of the gold nanoparticle agglomerates via endocytosis and co-resident molecules in the endosomes are released by applying laser irradiation, preventing their intraendosomal degradation. Due to the highly localized effect, the cell membrane integrity is not affected. Therefore, this technique can be an efficient tool for spatially and temporally confined intracellular release. The utilization of specifically designed photodispersible gold nanoparticle agglomerates (65 nm) can open novel avenues in imaging and molecule delivery. Due to the induced deagglomeration the primary, small particles (~5 nm) are more likely to be removed from the body.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0155-8) contains supplementary material, which is available to authorized users.
The fabrication of three-dimensional (3D) metal microstructures in a synthetic polymer-based hydrogel is demonstrated by femtosecond laser-induced photoreduction. The linear-shaped silver structure of approximately 2 micrometers in diameter is fabricated inside a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel. The silver structure is observed and confirmed by scanning electron microscopy (SEM) and elemental analysis using energy-dispersive X-ray spectroscopy (EDX). Shrinking and swelling of the fabricated structure is also demonstrated experimentally, which shows the potential of the present method for realizing 3D flexible electronic and optical devices, as well as for fabricating highly integrated devices at submicron scales.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.