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)

The cell selective introduction of therapeutic agents remains a challenging problem. Here we demonstrate spatially controlled cavitation instigated by laser-induced breakdown of an optically trapped single gold nanoparticle of diameter 100 nm. The energy breakdown threshold of the gold nanoparticle with a single nanosecond laser pulse at 532 nm is three orders of magnitude lower than water, which leads to nanocavitation allowing single cell transfection. We quantify the shear stress to cells from the expanding bubble and optimize the pressure to be in the range of 1-10 kPa for transfection. The transfection of genes and injection of therapeutic agents into individual mammalian cells are among the most important research tools in modern molecular biology [1]. The use of acoustic bubbles in the proximity of cells oscillated by ultrasonic irradiation (insonation) can lead to enhanced membrane permeabilization of cells, and is known as sonoporation. Acoustic streaming, shock waves, and liquid microjets associated with the dynamics of cavitation bubble are implicated in gene and drug delivery into cells [2]. This approach, however, often leads to nonuniform and sporadic molecular uptake that lacks cell selectivity and suffers from a significant loss of cell viability. Recently a suite of optical methods in the domain of cavitation-based therapies has provided the potential of sterility, reconfigurability, and single cell selectivity [3]. Laser-induced breakdown (LIB) of a liquid medium containing cells, has demonstrated cell lysis, necrosis or membrane permeabilization, with the outcome dependent upon the hydrodynamic shear stress to cells caused by the expanding bubble [4]. However, the relatively high energy deposition required for this process resulted in a much larger cavitation bubble (typically >200 μm in diameter) compared to the typical cell size that effectively reduces cell viability and has been detrimental to allow its wider usage.More spatially controlled cavitation may be achieved by optically trapping particles for subsequent LIB instead of the surrounding liquid [5]. Optical tweezers allow the positioning of individual nanoparticles or microparticles at a desired location within the buffer medium. Therefore, using this tool for LIB offers additional degrees of freedom-the particle material, its size, and the LIB position relative to cells or tissues. We have shown that the LIB of trapped polystyrene nanoparticles significantly reduced the energy required for cavitation [6,7]. This leads to the permeabilization of cell membranes and transfection of cells in a targeted area in the absence of a lysis zone of cells.Gold nanoparticle clusters have played a key role in this field, through antibody binding and sequestration with subsequent irradiation by nanosecond or femtosecond laser pulses [8][9][10]. A unique interaction of gold nanoparticles with light, known as surface plasmon resonance, can lead to strong absorption of light for heat generation. However, the introduction of gold nanoparticles in the c...

Willin/FRMD6 was first identified in the rat sciatic nerve, which is composed of neurons, Schwann cells, and fibroblasts. Willin is an upstream component of the Hippo signaling pathway, which results in the inactivation of the transcriptional co-activator YAP through Ser127 phosphorylation. This in turn suppresses the expression of genes involved in cell growth, proliferation and cancer development ensuring the control of organ size, cell contact inhibition and apoptosis. Here we show that in the mammalian sciatic nerve, Willin is predominantly expressed in fibroblasts and that Willin expression activates the Hippo signaling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These results show that Willin modulates sciatic nerve fibroblast activity indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.

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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.

We present a numerical technique for extended focused imaging and three-dimensional analysis of a microparticle field observed in a digital holographic microscope working in transmission. The three-dimensional localization of objects is performed using the local focus plane determination method based on the integrated amplitude modulus. We apply the refocusing criterion locally for each pixel, using small overlapping windows, to obtain the depth map and a synthetic image in which all objects are refocused independent from their refocusing distance. A successful application of this technique in the analysis of the microgravity particle flow experiment is presented.

We use Digital Holographic Microscopy to study dynamic responses of live cells to femtosecond laser cellular membrane photoporation. Temporal and spatial characteristics of morphological changes as well as dry mass variation are analyzed and compared with conventional fluorescent assays for viability and photoporation efficiency. With the latter, the results provide a new insight into the efficiency and toxicity of this novel optical method of drug delivery. In addition, quantitative phase maps reveal photoporation related sub-cellular dynamics of cytoplasmic vesicles.