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

Mulholland, W.; Kendall, M. A. F.

doi:10.1117/12.516059

Cited by 8 publications

(7 citation statements)

References 19 publications

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“…We used a modified femtosecond two‐/multiphoton laser scanning system (Mulholland, 2004) configured for the non‐invasive 3D imaging of thick biological specimens as recently described and detailed by Tirlapur et al. (2006).…”

Section: Methodsmentioning

confidence: 99%

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

et al. 2007

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The distribution of microfibrils was studied immunohistochemically in intervertebral discs taken from young normal human surgical cases and from the bovine tail. Co-localization of microfibrils and elastin fibres was examined by dual immunostaining of fibrillin-1 and elastin. Collagen fibre network orientation was studied by using polarized filters. A similar microfibrillar network was seen in both bovine and human discs with network organization being completely different from region to region. In the outer annulus fibrosus (OAF), abundant microfibrils organized in bundles were mainly distributed in the interterritorial matrix. In addition, the microfibril bundles were orientated parallel to each other and co-localized highly with elastin fibres. Within each lamella, co-localized microfibrils and elastin fibres were aligned in the same direction as the collagen fibres. In the interlamellar space, a dense co-localized network, staining for both microfibrils and elastin fibres, was apparent; immunostaining for both molecules was noticeably stronger than within lamellae. In the inner annulus fibrosus, the microfibrils were predominantly visible as a filamentous mesh network, both in the interterritorial matrix and also around the cells. The microfibrils in this region co-localized with elastin fibres far less than in the OAF. In nucleus pulposus, filamentous microfibrils were organized mainly around the cells where elastin fibres were hardly detected. By contrast, sparse elastin fibres, with a few of microfibrils, were visible in the interterritorial matrix . The results of this study suggest the microfibrillar network of the annulus may play a mechanical role while that around the cells of the nucleus may be more involved in regulating cell function.

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Section: Methodsmentioning

confidence: 99%

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

et al. 2007

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“…A diode pumped Ti:Sapphire crystal laser (Mira-Coherent, Ely, UK) provided 150 fs pulses of NIR light, selectively tunable between 700 and 980 nm. The beam was aligned, attenuated, and scanned across the sample using a directly coupled multiphoton system described previously (Mulholland and Kendall, 2004). Briefly, it consisted of a BioRad Radiance 2100 MPD laser-scanning and control system (Bio-Rad Microsciences Ltd., now Carl Zeiss GmbH, Jena, Germany) coupled to a Nikon E600 FN upright microscope (Nikon UK Ltd, Surrey, UK).…”

Section: Mplsm Systemmentioning

confidence: 99%

Multiphoton High-Resolution 3D Imaging of Langerhans Cells and Keratinocytes in the Mouse Skin Model Adopted for Epidermal Powdered Immunization

Mulholland

Arbuthnott

Bellhouse

et al. 2006

Journal of Investigative Dermatology

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Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 microm, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 microm3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

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“…and Microspectroscopy We used a modified femtosecond two/multiphoton laser scanning system (configured for the noninvasive 3D imaging of skin in vivo) previously described by Mulholland and Kendall (2004). A schematic of the major components of the TPLSM system is presented in Figure 1.…”

Section: Two-photon Immunofluorescence Imagingmentioning

confidence: 99%

Femtosecond two‐photon high‐resolution 3D imaging, spatial‐volume rendering and microspectral characterization of immunolocalized MHC‐II and mLangerin/CD207 antigens in the mouse epidermis

Tirlapur¹,

Mulholland²,

Bellhouse³

et al. 2006

Microscopy Res & Technique

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Langerhans cells (LCs) play a sentinel role by initiating both adaptive and innate immune responses to antigens pertinent to the skin. With the discovery of various LCs markers including antibodies to major histocompatibility complex class II (MHC-II) molecules and CD1a, intracellular presence of racket-shaped "Birbeck granules," and very recently Langerin/CD207, LCs can be readily distinguished from other subsets of dendritic cells. Femtosecond two-photon laser scanning microscopy (TPLSM) in recent years has emerged as an alternative to the single photon-excitation based confocal laser scanning microscope (CLSM), particularly for minimally-invasive deep-tissue 3D and 4D vital as well as nonvital biomedical imaging. We have recently combined high resolution two-photon immunofluorescence (using anti MHC-II and Langerin/CD207 antibodies) imaging with microspectroscopy and advanced image-processing/volume-rendering modalities. In this work, we demonstrate the use of this novel state-of-the-art combinational approach to characterize the steady state 3D organization and spectral features of the mouse epidermis, particularly to identify the spatial distribution of LCs. Our findings provide unequivocal direct evidence that, in the mouse epidermis, the MHC-II and mLangerin/CD207 antigens do indeed manifest a high degree of colocalization around the nucleus of the LCs, while in the distal dendritic processes, mLangerin/CD207 antigens are rather sparsely distributed as punctuate structures. This unique possibility to simultaneously visualize high resolution 3D-resolved spatial distributions of two different immuno-reactive antigens, namely MHC-II and mLangerin/CD207, along with the nuclei of LCs and the adjacent epidermal cells can find interesting applications. These could involve aspects associated with pragmatic analysis of the kinetics of LCs migration as a function of immuno-dermatological responses during (1) human Immunodeficiency virus disease progression, (2) vaccination and targeted gene therapy, (3) skin transplantation/plastic surgery, (4) ultraviolet and other radiation exposure, (5) tissue-engineering of 3D skin constructs, as well as in (6) cosmetic industry, to unravel the influence of cosmeceuticals.

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

Cited by 8 publications

References 19 publications

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

Multiphoton High-Resolution 3D Imaging of Langerhans Cells and Keratinocytes in the Mouse Skin Model Adopted for Epidermal Powdered Immunization

Femtosecond two‐photon high‐resolution 3D imaging, spatial‐volume rendering and microspectral characterization of immunolocalized MHC‐II and mLangerin/CD207 antigens in the mouse epidermis

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