Mr. William J. Mulholland Profile

William J. Mulholland

Research Student at Trinity College-Univ of Melbourne

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Proceedings Article | 18 February 2004 Paper

Design and commissioning of a directly coupled in-vivo multiphoton microscope for skin imaging in humans and large animals

William Mulholland, Mark Kendall

Proceedings Volume 5249, (2004) https://doi.org/10.1117/12.516059

KEYWORDS: Microscopes, Imaging systems, In vivo imaging, Skin, Objectives, Luminescence, Near infrared, Sensors, Animal model studies, Head

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The application of near infrared multiphoton excitation to the laser-scanning microscope was first conceived by Denk, Strickler and Webb in 1990. Since then, advances in design have seen the multiphoton laser scanning microscope (MPLSM) applied to a wide range of biological research areas, including skin imaging and vaccine delivery. The technique has the attributes of low phototoxicity, high-resolution functional imaging to depths in scattered tissues. These characteristics have encouraged engineers and scientists to develop in-vivo imaging systems. For these applications, laser excitation pulses can be delivered to the sample through optical fibers. Although this solution provides a number of advantages relating to movement and flexibility of the site of interest relative to the laser source, the peak powers that can be delivered down the fiber are limited. We report on the design and commissioning of a directly coupled in-vivo MPM system, optimised for the imaging of epidermal vaccines delivered to a range of biological models and humans. Specifically, we seek to apply the system to visualise in-vivo, the influence of hand-held, helium powered needle-free systems on skin cells. A standard Nikon E600FN microscope, dissected above the optical plane was cantilevered from a vibration isolated table using rigid support arms. The modified microscope was coupled to an infrared optimised Bio-Rad Radiance 2100MP, multiphoton dedicated laser scanning control and image acquisition system. Femtosecond laser pulses were provided by a 10W Verdi pumped Mira Ti:Sapphire laser, from Coherent Inc. The microscope was modified such that the transmission half may be selectively attached for conventional imaging with ex-vivo and cell culture samples, or removed for in-vivo imaging of skin sites on the body of humans and large animals. Optical performance of the system, and aspects of its design and commissioning are discussed in this paper.

Proceedings Article | 17 June 2002 Paper

Analysis of microparticle penetration into human and porcine skin: non-invasive imaging with multiphoton excitation microscopy

William Mulholland, Mark Kendall, Brian Bellhouse, Nick White

Proceedings Volume 4620, (2002) https://doi.org/10.1117/12.470685

KEYWORDS: Skin, Particles, Image processing, Multiphoton microscopy, Microscopy, Tissues, Gold, Image resolution, Ultraviolet radiation, Photonic integrated circuits

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At the University of Oxford and PowderJect Pharmaceuticals plc, a unique form of needle-free injection technology has been developed. Powdered vaccines and drugs in micro-particle form are accelerated in a high-speed gas flow to sufficient velocity to enter the skin, subsequently achieving a pharmaceutical effect. To optimize the delivery of vaccines and drugs with this method a detailed understanding of the interactive processes that occur between the microparticles and the skin is necessary. Investigations to date of micro-particle delivery into excised human and animal tissue have involved image analyses of histology sections. In the present study, a series of investigations were conducted on excised human and porcine skin using the technique of Multi-Photon fluorescence excitation Microscopy (MPM) to image particles and skin structures post-penetration. Micro-particles of various size and composition were imaged with infrared laser excitation. Three-dimensional images of stratum corneum and epidermal cell deformation due to micro-particle penetration were obtained. Measurements of micro-particle penetration depth taken from z-scan image stacks were used to successfully quantify micro-particle distribution within the skin, without invasively disrupting the skin target. This study has shown that MPM has great potential for the non-invasive imaging of particle skin interactive processes that occur with the transdermal delivery of powdered micro-particle vaccines and drugs.

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