Maria Leilani Torres-Mapa

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.

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.

Spatially optimized gene transfection by laser-induced breakdown of optically trapped nanoparticles

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.

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.

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.

Transient transfection of mammalian cells using a violet diode laser

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.Photoporation is the use of tightly focused laser light to perforate the cellular membrane and allow exogenous material to be taken up by the cell [1]. This technique has become increasingly popular due to its simplicity, robustness and efficiency. Most of its applications are predominantly, but not limited to, the delivery of nucleic acids such as plasmid DNA and messenger RNA to intracellular compartments. Aside from genetic material, dyes, nanoparticles and possibly quantum dots can also be injected to the cells which can be useful for monitoring gene or drug activity.

Femtosecond laser direct writing of metal microstructure in a stretchable poly(ethylene glycol) diacrylate (PEGDA) hydrogel

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.

Shrinkable silver diffraction grating fabricated inside a hydrogel using 522-nm femtosecond laser

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.

Gold nanoparticle-mediated delivery of molecules into primary human gingival fibroblasts using ns-laser pulses: A pilot study

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.

Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

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.