The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture.

Photodynamic therapy (PDT) is an alternative cancer treatment to conventional surgery, radiotherapy and chemotherapy. It is based on activating a drug with light that triggers the generation of cytotoxic species that promote tumour cell killing. At present, PDT is mainly used in the treatment of wet age-related macular degeneration, for precancerous conditions of the skin (e.g. actinic keratosis) and in the palliative care of advanced cancers, for instance of the bladder or the oesophagus. Due to a lack of phase III clinical trials and limitations of light penetration to deeper lying tumours (in excess of 1 cm), PDT is still not used as a first line cancer treatment, which is surprising given the first clinical trials by Dougherty's group dating back to the 1970's. However research continues to demonstrate the potential benefits of PDT and the need to stimulate funding and uptake of clinical studies using next generation photosensitizers offering advanced targeted delivery, improved photodynamic dose combined with modern light delivery technologies. This review surveys the available PDT treatments and emerging novel developments in the field with a particular focus on two-photon techniques that are anticipated to improve the effectiveness of PDT in tissues at depth and on next generation drugs that work without the need of the presence of oxygen for photosensitization making them effective where hypoxia has taken hold.

To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of Ecombretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to 2 orders of magnitude higher than that of the drug in the surrounding medium.

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low- and high-molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one- or two-colour lasers avoids the need for ultraviolet-transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E-C1 or E-C2) for versatile use with Ti-sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.

Abstract. The photoisomerization of relatively non-toxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one-and two-photon excitations at 340 and 625 nm respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygenindependent and active in hypoxic environments such as in tumors. The use of red or near-infrared light for twophoton excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes non-discriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of 2-photon red or near-infrared (NIR) excitation for deeper tissue penetration.

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Abstract. The uptake of E -combretastatins, potential prodrugs of the anticancer Z -isomers, into multicellular spheroids has been imaged by intrinsic fluorescence in three dimensions using two-photon excited fluorescence lifetime imaging with 625-nm ultrafast femtosecond laser pulses. Uptake is initially observed at the spheroid periphery but extends to the spheroid core within 30 min. Using agarose gels as a three-dimensional model, the conversion of Z ðtransÞ → E ðcisÞ via two-photon photoisomerization is demonstrated and the location of this photochemical process may be precisely selected within the micron scale in all three dimensions at depths up to almost 2 mm. We discuss these results for enhanced tissue penetration at longer near-infrared wavelengths for cancer therapy and up to three-photon excitation and imaging using 930-nm laser pulses with suitable combretastatin analogs. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

This study outlines a novel manufacturing technique for the generation of compartmentalized trilayered nanoparticles loaded with an anti-cancer agent and siRNA as a platform for the combination treatment of cancers. More specifically, we describe the use of a multi-needle electrohydrodynamic approach to produce nanoparticles with high size specificity and scalable output, while allowing suitable environments for each therapeutic agent. The inner polylactic-glycolic-acid (PLGA) layer was loaded with cisplatin while the middle chitosan layer was loaded with siRNA. The corresponding polymeric solutions were characterized for their viscosity, surface tension and conductivity, while particle size was determined using dynamic light scattering. The internal structure was studied using transmission electron microscopy (TEM) and Structured Illumination Microscopy (SIM). The inclusion of cisplatin was studied using electron dispersive spectroscopy (EDS). We were able to generate nanoparticles of approximate size 130 nm with three distinct layers containing an outer protective PLGA layer, a middle layer of siRNA and an inner layer of cisplatin. These particles have the potential not only for uptake into tumors via the enhanced permeability and retention (EPR) effect but also the sequential release of the siRNA and chemotherapeutic agent, thereby providing a means of overcoming challenges of targeting and tumor drug resistance.