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All optical approaches to control and read out the electrical activity in a cardiac syncytium can improve our understanding of cardiac electrophysiology. Here, we demonstrate optogenetic stimulation of cardiomyocytes with high spatial precision using light foci generated with a ferroelectric spatial light modulator. Computer generated holograms binarized by bidirectional error diffusion create multiple foci with more even intensity distribution compared with thresholding approach. We evoke the electrical activity of cardiac HL1 cells expressing the channelrhodopsin-2 variant, ChR2(H134R) using single and multiple light foci and at the same time visualize the action potential using a calcium sensitive indicator called Cal-630. We show that localized regions in the cardiac monolayer can be stimulated enabling us to initiate signal propagation from a precise location. Furthermore, we demonstrate that probing the cardiac cells with multiple light foci enhances the excitability of the cardiac network. This approach opens new applications in manipulating and visualizing the electrical activity in a cardiac syncytium.

Microfluidic systems facilitate the realization of compact and miniaturized lab-on-a-chip systems which can be used for various applications. The conventional method to fabricate such devices entails the use of complex etching processes in clean rooms and soft lithography methods which require substantial expertise. Since the design of such microfluidic devices is often customized depending on the application of the user, it would be ideal to have a fabrication technique that would allow for fast and reliable production with the possibility to generate high resolution three-dimensional structures using different materials. 3D printing technique has been recently demonstrated as a means to fabricate microfluidic devices. It enables rapid prototyping of robust and complex structures. Nowadays, 3D printers can create small structures down to several tens of microns. 3D-printed devices can also provide lab-on-a-chip systems which are compatible with optical techniques and microscopy. In this work, we demonstrate the integration of optical manipulation in 3D-printed microfluidic systems with particular focus on optimized design and fabrication protocol. 3D printing was performed using a Multijet printer (MJP2500 Plus). A microfluidic chip was designed for the purpose of dual beam optical trapping and optical stretching of mammalian cells. Three inlets with channel dimensions of 500 µm were used to flow buffer, particles or cells into the device. Two single mode fibers were inserted into fiber guide channels with dimensions of 500 µm, separated with a distance of 300 µm, in order to deliver counterpropagating beams into the trapping region. Hydrodynamic focusing was performed showing that laminar flow can be achieved in the device. In order to evaluate the compatibility of 3D-printed microfluidic chips for optical manipulation, the mean square displacement of the optically trapped 10 µm polystyrene particle was measured for different laser powers. In addition, we demonstrate optical stretching4 of microvascular endothelial cells under flow.

On cellular or tissue level, electrophysiology studies during light stimulation with shaped light can potentially enable the design of effective stimulation strategies towards future cardiac therapies. In this work, we explore the use of optogenetics in probing the electrophysiology of a functional cardiac syncytium. A holographic technique with the use of a spatial light modulator allows beam shaping and precise spatiotemporal control of the origin of action potential in a cardiac-like cell line monolayer. Depolarization was performed by using a blue laser (488 nm) incident to a reflective ferroelectric liquid crystal on silicon spatial light modulator (FLCOS-SLM) which generates multiple foci on to cardiomyocyte-like cells (HL1 line) expressing the light-gated protein Channelrhodopsin-2 (ChR2). The calcium-sensitive fluorophore Cal-630 was used to visualize the cellular electrical activity and thus can be combined with ChR2 as an all-optical method to stimulate and visualize electrophysiology of cardiac-like cells in a non-contact manner. We generated single or multiple foci each with spot size of ~5 µm for optogenetic stimulation. The probability of triggering an action potential, which we refer to as excitation probability, is highly dependent on the laser intensity. The threshold intensity for a single beamlet, where approximately 50% of the laser pulses successfully trigger an action potential, is ~4 µW/μm² (n=5 regions, 5 cells analyzed per region, performed in triplicates) at frequency of 1 Hz and pulse duration of 10 ms. We also demonstrate spatial summation of membrane potential in HL1-ChR2. Generated multiple foci can render the excited cells as individual inputs to elicit action potential in a syncytium. We demonstrate the dependence of the excitation probability on the foci gap distance. Two foci, each with sub-threshold intensity of 2 µW/μm² were generated. At spacing of 50 µm, the median excitation probability increases compared to single spot stimulation with the same intensity.

In the past decade, in vivo models for optogenetic applications have gained importance, especially in the fields of cardiology and neuroscience. To reliably evoke the desired reactions while minimizing adverse effects, the stimulation power must be carefully adjusted. The relevant light intensity for cells in deeper layers of scattering tissue is not easily extrapolated from the power threshold of single cells. In this study, we evaluated a model for deep tissue optogenetic stimulation, using a heart-like cell line and tissue phantoms. Phantoms were fabricated from PDMS and titanium dioxide particles and possessed highly reproducible optical properties. Scattering and absorption coefficients were modeled to match those of realistic tissues. Since power of light traveling through tissues decays exponentially with respect to the scattering and absorption coefficients, the required input power was expected to increase exponentially by the same factor. To test this hypothesis, cells were stimulated through tissue phantoms of varying thickness with different modes of illumination. Cellular reactions revealed that the simplified assumptions were not sufficient to predict the input power required to reach the stimulation threshold. We provide a more comprehensive model to assess cellular reactions in scattering tissues a priori. This study has implications for the use of optogenetics in tissue models, organs and in vivo models as the outcomes can be transferred to different types of cells and tissues.

The integration of metal microstructures and soft materials is promising for the realization of novel optical and biomedical devices owing to the flexibility and biocompatibility of the latter. Nevertheless, the fabrication of three-dimensional metal structures within a soft material is still challenging. In this study, we demonstrate the fabrication of a silver diffraction grating inside a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel by using a 522-nm femtosecond laser via multi-photon photoreduction of silver ions. The optical diffraction pattern obtained with the grating showed equally spaced diffraction spots, which indicated that a regular, periodic silver grating was formed. Notably, the distance between the diffraction spots changed when the water content in the hydrogel was reduced. The grating period decreased when the hydrogel shrank owing to the loss of water, but the straight shapes of the line structures were preserved, which demonstrated the optical tunability of the fabricated structure. Our results demonstrate the potential of the femtosecond laser-based photoreduction technique for the fabrication of novel tunable optical devices as well as highly precise structures.

Advances in photomedicine and optogenetics have defined the problem of efficient light delivery in vivo. Recently, hydrogels have been proposed as alternatives to glass or polymer fibers. These materials provide remarkable versatility, biocompatibility and easy fabrication protocols. Here, we investigate the usability of waveguides from poly(ethylene glycol) dimethacrylate for targeted light delivery and diffusion. Different hydrogel compositions were characterized with regard to water content, chemical stability, elasticity, refractive index and optical losses. Differences in refractive index were introduced to achieve targeted light delivery, and scattering polystyrene particles were dispersed in the hydrogel samples to diffuse the incident light. Complex constructs were produced to demonstrate the versatility of hydrogel waveguides. © 2019 Optical Society of America.

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day-old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225 mJ/cm2 induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser-based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

Light as a tool in medical therapy and biological research has been studied extensively and its application is subject to continuous improvement. However, safe and efficient application of light-based methods in photomedicine or optogenetics requires knowledge about the optical properties of the target tissue as well as the response characteristics of the stimulated cells. Here, we used tissue phantoms and a heart-like light-sensitive cell line to investigate optogenetic stimulation through tissue layers. The input power necessary for successful stimulation could be described as a function of phantom thickness. A model of light transmission through the tissue phantoms gives insights into the expected stimulation efficiency. Cell-type specific effects are identified that result in deviations of the stimulation threshold from the modelled predictions. This study provides insights into the complex interplay between light, tissue and cells during deep-tissue optogenetics. It can serve as an orientation for safe implementation of light-based methods in vivo.

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.

Interaction of gold nanoparticles (AuNPs) in the vicinity of cells' membrane with a pulsed laser (λ = 532 nm, τ = 1 ns) leads to perforation of the cell membrane, thereby allowing extracellular molecules to diffuse into the cell. The objective of this study was to develop an experimental setting to deliver molecules into primary human gingival fibroblasts (pHFIB-G) by using ns-laser pulses interacting with AuNPs (study group). To compare the parameters required for manipulation of pHFIB-G with those needed for cell lines, a canine pleomorphic adenoma cell line (ZMTH3) was used (control group). Non-laser-treated cells incubated with AuNPs and the delivery molecules served as negative control. Laser irradiation (up to 35 mJ/cm2) resulted in a significant proportion of manipulated fibroblasts (up to 85%, compared to non-irradiated cells: p < 0.05), while cell viability (97%) was not reduced significantly. pHFIB-G were perforated as efficiently as ZMTH3. No significant decrease of metabolic cell activity was observed up to 72 h after laser treatment. The fibroblasts took up dextrans with molecular weights up to 500 kDa. Interaction of AuNPs and a pulsed laser beam yields a spatially selective technique for manipulation of even primary cells such as pHFIB-G in high throughput. © 2016 by the authors.

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.

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)

This work is supported by the UK Engineering Physical Sciences Research Council (EPSRC). 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. Publisher PDF

Summary form only given. Optical techniques such as poration and trapping can be viable tools toward micromanipulation of living organisms. In embryos, there are two major technical issues that need to be addressed. Injection of fluorophores or genetic constructs into a single cell of an embryo will enable cell-fate mapping and determine timing dependent protein functionality during its development. Furthermore, a non-contact method for orienting the embryo at its later stage of development is desirable for high magnification imaging. In this work, we present a holographic based system that can be utilized for both approaches as shown in our previous work [1]. Using a pulsed Ti:Sapphire femtosecond (fs) laser and a spatial light modulator (SLM), we can demonstrate successful injection of impermeable molecules such as fluorescently labeled dextran molecules and Propidium iodide into Pomatoceros lamarckii embryos. By using the continuous wave output of a Ti:Sapphire laser, we can perform optical trapping of embryos using low numerical aperture (NA) objectives.We demonstrate optical poration of developing embryos in its early stages of development with dextran molecules of varying molecular weights, from 3 kDa to 500 kDa. A single blastomere can be optoinjected with a tightly focused pulsed Ti:Sapphire fs laser shown in Fig.1(A). We characterized the optical poration efficiency by bathing the embryos in a sea water solution mixed with 10 μM of Propidium iodide. This dye was chosen so as to avoid the necessity of washing the embryos after optical poration. The efficiency ranged from 44% to 55% for early-stage to more developed embryos by sequential delivery of fs pulses targeting at three different spatial positions as demonstrated in our previous work [2] using laser power of 65 mW and exposure time of 30 ms. The holographic system presented can also be used to optically orient or trap a developing embryo. By wavefront modulation of the incident beam at the Fourier plane, the beam can be positioned laterally and displaced axially. We show that we can optically trap an embryo using a low NA, 20x objective as shown in Fig.1(B). The embryo rotates about an axis during trapping. By producing a dual focus trap using the SLM created by superposing overlapping foci, 36 μm apart, we can stably trap and fixed the orientation of a singlecell embryo.

We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

Transgenic animals are an essential means for investigating genetic processes in vivo, and depend on efficient delivery techniques to introduce exogenous genetic material into the organism, often at the zygote stage. In this study, we demonstrate an optical approach to microinjection based on a holographic system using a spatial light modulator and a Ti: Sapphire laser. This integrated system is capable of both optical orientation and injection of 60- μm diameter Pomatoceros lamarckii (P.lamarckii) embryos. Individual blastomeres of P. lamarckii embryos were optoinjected with varying sizes of dextran molecules and Propidium iodide using an 800-nm femtosecond laser with controlled dosage. We also show that the technique is able to deliver materials to cells located deep within a welldeveloped embryo. As a visual confirmation of successful optoinjection, the presence of gas bubbles was observed as a function of laser power and exposure time. Small gas bubbles, less than 5-μm in diameter, were found to be tolerated by the irradiated embryo. Furthermore, when switched to the continuous wave mode, the laser could exert optical forces upon the embryo. This facilitated computer-controlled handling and orientation of P. lamarckii embryos without compromising viability. Our multimodal optical platform offers a sterile, non-contact and robust alternative to traditional microinjection. This work is a step towards applications in developmental biology such as cell lineage mapping and formation of transgenic animals using an optical approach.

Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

The Salvador/Warts/Hippo (Hippo) pathway defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Hippo pathway was initially utilised in D. melanogaster, where the Expanded protein acts in the Hippo signalling cascade to control organ size. Willin is the proposed human orthologue of Expanded and the aim of this thesis is to investigate whether willin can activate the mammalian Hippo signalling pathway. Ectopic willin expression causes an increase in phosphorylation of the core Hippo signalling pathway components MST1/2, LATS1 and YAP, an effect which can be antagonised by ezrin. In MCF10A cells, willin over-expression antagonises a YAP-induced epithelial-to-mesenchymal transition via the N- terminal FERM (Four-point-one Ezrin Radixin Moesin) domain of willin. Preliminary results show that willin is expressed within the sciatic nerve of rat and mice, and within the neuromast cells in the zebrafish; suggesting that willin and the Hippo pathway may play a vital role in the developmental regulation within the peripheral nervous system. To conclude, willin influences Hippo signalling activity by activating the core Hippo pathway kinase cassette in mammalian cells.

A novel approach to gene transfection is demonstrated. It uses laser-induced breakdown of an optically trapped single nanoparticle to achieve a high transfection efficiency in a quasi-targeted manner, without cell lysis, using a nanosecond laser.

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. Publisher PDF

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. Publisher PDF

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.

Background Cell-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. Results Gold 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 (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda$$\end{document}λ = 532 nm, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{pulse}$$\end{document}τpulse = 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\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{2}$$\end{document}2 and above. Successful intracellular release was shown using the fluorescent dye calcein. For a radiant exposure of 35 mJ/cm\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{2}$$\end{document}2 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. Conclusion CPPs 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 material The online version of this article (doi:10.1186/s12951-015-0155-8) contains supplementary material, which is available to authorized users.