VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy.

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.

New Approaches to Photodynamic Therapy from Types I, II and III to Type IV Using One or More Photons.

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.

Three-dimensional imaging and uptake of the anticancer drug combretastatin in cell spheroids and photoisomerization in gels with multiphoton excitation.

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(trans)→E(cis) 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.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution.

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5-50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.

Nanometric molecular separation measurements by single molecule photobleaching.

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging.

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.

Anticancer phototherapy using activation of E-combretastatins by two-photon-induced isomerization.

The photoisomerization of relatively nontoxic 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 oxygen-independent and active in hypoxic environments such as in tumors.

Ultrafast vibrational spectroscopic studies on the photoionization of the α-tocopherol analogue trolox C.

The initial events after photoexcitation and photoionization of α-tocopherol (vitamin E) and the analogue Trolox C have been studied by femtosecond stimulated Raman spectroscopy, transient absorption spectroscopy and time-resolved infrared spectroscopy. Using these techniques it was possible to follow the formation and decay of the excited state, neutral and radical cation radicals and the hydrated electron that are produced under the various conditions examined. α-Tocopherol and Trolox C in methanol solution appear to undergo efficient homolytic dissociation of the phenolic -OH bond to directly produce the tocopheroxyl radical.

Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells.

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.