All-Optical Assay to Study Biological Neural Networks.

We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally, we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.

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.

Inhibition of Plk1 and Cyclin B1 expression results in panobinostat-induced G2 delay and mitotic defects.

The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G2/M and an associated decrease in expression of particular genes required for passage through G2 and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G2 delay and mitotic defects. A loss of G2-specific Plk1 and Cyclin B1 expression and co-incident increase in p21(Waf1/Cip1) expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC.

Laser-induced breakdown of an optically trapped gold nanoparticle for single cell transfection.

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 method shows transfection of plasmid DNA into individual mammalian cells with an efficiency of 75%.

Femtosecond optical transfection of individual mammalian cells.

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.

Willin, an upstream component of the hippo signaling pathway, orchestrates mammalian peripheral nerve fibroblasts.

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.

Exploring the ultrashort pulse laser parameter space for membrane permeabilisation in mammalian cells.

The use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.

Multimodal biophotonic workstation for live cell analysis.

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy.

Imaging the cellular response to transient shear stress using stroboscopic digital holography.

We use stroboscopic quantitative phase microscopy to study cell deformation and the response to cavitation bubbles and transient shear stress resulting from laser-induced breakdown of an optically trapped nanoparticle. A bi-directional transient displacement of cytoplasm is observed during expansion and collapse of the cavitation bubble. In some cases, cell deformation is only observable at the microsecond time scale without any permanent change in cell shape or optical thickness. On a time scale of seconds, the cellular response to shear stress and cytoplasm deformation typically leads to retraction of the cellular edge most exposed to the flow, rounding of the cell body and, in some cases, loss of cellular dry mass. These results give a new insight into the cellular response to cavitation induced shear stress and related plasma membrane permeabilization. This study also demonstrates that laser-induced breakdown of a nanoparticle offers localized cavitation, which interacts with a single cell but without causing cell lysis.

Integrated holographic system for all-optical manipulation of developing embryos.

We demonstrate a system for the combined optical injection and trapping of developing embryos. A Ti:sapphire femtosecond laser in tandem with a spatial light modulator, is used to perform fast and accurate beam-steering and multiplexing. We show successful intracellular delivery of a range of impermeable molecules into individual blastomeres of the annelid Pomatoceros lamarckii embryo by optoinjection, even when the embryo is still enclosed in a chorion. We also demonstrate the ability of the femtosecond laser optoinjection to deliver materials into inner layers of cells in a well-developed embryo. By switching to the continuous wave mode of the Ti:sapphire laser, the same system can be employed to optically trap and orient the 60 µm sized P. lamarckii embryo whilst maintaining its viability. Hence, a complete all-optical manipulation platform is demonstrated paving the way towards single-cell genetic modification and cell lineage mapping in emerging developmental biology model species.

Enhancement and optimization of plasmid expression in femtosecond optical transfection.

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non-invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3-fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO-K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ~70% without compromising the transfection efficiency. Also, we report for the first time the successful photo-transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices.

Application of dynamic diffractive optics for enhanced femtosecond laser based cell transfection.

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.

Quantitative phase study of the dynamic cellular response in femtosecond laser photoporation.

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.

Extended focused imaging of a microparticle field with digital holographic microscopy.

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.