Publications 2011

Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels

Patalay R, Talbot C, Alexandrov Y, Munro I, Neil MA, König K, French PM, Chu A, Stamp GW, Dunsby C.

Biomed Opt Express. 2011 Dec 1;2(12):3295-308. doi: 10.1364/BOE.2.003295. Epub 2011 Nov 10

Abstract:

We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed.

 

Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research

König K, Raphael AP, Lin L, Grice JE, Soyer HP, Breunig HG, Roberts MS, Prow TW.

Adv Drug Deliv Rev. 2011 Apr 30;63(4-5):388-404. doi: 10.1016/j.addr.2011.03.002. Epub 2011 Apr 13.

Abstract:

Multiphoton tomography for in vivo high-resolution multidimensional imaging has been used in clinical investigations and small animal studies. The novel femtosecond laser tomographs have been employed to detect cosmetics and pharmaceutical components in situ as well as to study the interaction of drugs with intratissue cells and the extracellular matrix under physiological conditions. Applications include the intra-tissue accumulation of sunscreen nanoparticles in humans, the monitoring the metabolic status of patients with dermatitis, the biosynthesis of collagen after administration of anti-aging products, and the detection of porphyrins after application of 5-aminolevulinic acid. More than 2000 patients and volunteers in Europe, Australia, and Asia have been investigated with these unique tomographs. In addition, femtosecond laser nanoprocessing microscopes have been employed for targeted delivery and deposition in body organs, optical transfection and optical cleaning of stem cells, as well as for the optical transfer of molecular beacons to track microRNAs. These diverse applications highlight the capacity for multiphoton tomography and femtosecond laser nanoprocessing tools to advance drug delivery research.

 

Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging

Talbot CB, Patalay R, Munro I, Warren S, Ratto F, Matteini P, Pini R, Breunig HG, König K, Chu AC, Stamp GW, Neil MA, French PM, Dunsby C.

Opt Express. 2011 Jul 18;19(15):13848-61. doi: 10.1364/OE.19.013848.

Abstract:

When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.

 

Quantification of cellular autofluorescence of human skin using  multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels

Rakesh Patalay, Clifford Talbot, Yuriy Alexandrov, Ian Munro, Mark A. A. Neil, Karsten König, Paul M. W. French, Anthony Chu, Gordon W. Stamp, and Chris Dunsby

Biomed Opt Express. 2011 December 1; 2(12): 3295–3308.

Abstract:

We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed.

 

Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy

Benati E, Bellini V, Borsari S, Dunsby C, Ferrari C, French P, Guanti M, Guardoli D, Koenig K, Pellacani G, Ponti G, Schianchi S, Talbot C, Seidenari S.

Skin Res Technol. 2011 Apr 25. doi: 10.1111/j.1600-0846.2011.00496.x.

Abstract:

Background/purpose: Multiphoton microscopy (MPM) enables the assessment of unstained living biological tissue with submicron resolution, whereas fluorescence lifetime imaging microscopy (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. The aim of this study was to compare the healthy skin of young individuals with that of older subjects, as well as to assess the skin at different body sites, by means of MPM and FLIM. Methods: Nineteen elderly patients were examined on the outer side of the forearm, whereas 30 young individuals were assessed on the dorsal and volar sides of the forearm and on the thigh. Results: Cell and nucleus diameters, cell density and FLIM vary according to the epidermal cell depth and the skin site. In elderly subjects, epidermal cells show morphologic alterations in shape and size, with smaller cell and nucleus diameters; the number of basal cells is decreased, whereas the mean fluorescence lifetimes at both the upper and the lower layers increase. Conclusion: This study provides quantitative and qualitative data on normal epidermis at different skin sites at different ages and represents a reference for the clinician attempting to understand the effectiveness of MPM and FLIM in discriminating diseased states of the skin from normal ones.

 

Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

Roberts MS, Dancik Y, Prow TW, Thorling CA, Lin LL, Grice JE, Robertson TA, König K, Becker W.

Eur J Pharm Biopharm. 2011 Apr;77(3):469-88. doi: 10.1016/j.ejpb.2010.12.023. Epub 2011 Jan 21.

Abstract:

New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.

 

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation

König K, Uchugonova A, Gorjup E.

Microsc Res Tech. 2011 Jan;74(1):9-17. doi: 10.1002/jemt.20866.

Abstract:

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.

 

Optical skin biopsies by clinical CARS and multiphoton fluorescence/SHG tomography

K. König, H.G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, E. Büttner, W. Sterry, J. Lademann

Laser Physics Letters  Volume 8, Issue 6, pages 465–468, June 2011

Abstract:

The ultimate challenge for early diagnostics is labelfree high-resolution intratissue imaging without taking physical biopsies. A novel hybrid femtosecond laser tomograph provides in vivo optical biopsies of human skin based on non-linear excitation of autofluorescence and the detection of lipids and water by CARS. Applications include skin cancer detection, biosafety tests of intradermal nanoparticles, and the testing of anti-aging products.

 

Two-color Raman spectroscopy for the simultaneous detection of chemotherapeutics and antioxidative status of human skin

W. Werncke, I. Latka, S. Sassning, B. Dietzek, M.E. Darvin, M.C. Meinke, J. Popp, K. König, J.W. Fluhr, J. Lademann

Laser Physics Letters  Volume 8, Issue 12, pages 895–900, December 2011

Abstract:

Aiming at the development of strategies to prevent the hand-foot-syndrome, we propose to evaluate the amount of chemotherapeutics in the human skin together with carotenoids the latter serving as marker substances for the dermal antioxidative status. This approach is demonstrated by applying two-color Raman spectroscopy at 785 and 532 nm excitation for selective detection of chemotherapeutics and carotenoids, respectively. Porcine ear skin has proven to be suited as a model for corresponding spectroscopic basic in-vitro investigations.

 

Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases

Koehler MJ, Speicher M, Lange-Asschenfeldt S, Stockfleth E, Metz S, Elsner P, Kaatz M, König K.

Exp Dermatol. 2011 Jul;20(7):589-94. doi: 10.1111/j.1600-0625.2011.01279.x. Epub 2011 May 4.

Abstract:

Multiphoton tomography (MPT) is an in vivo imaging technique with very high spatial resolution and efforts are made to combine MPT with other non-invasive imaging methods. The goals of the present study were the description of the features of different dermatological entities as seen in MPT and confocal laser scanning microscopy (CLSM) comparison of these two novel techniques and the 'classical' diagnostic measures visual inspection, dermoscopy and histology with respect to the strengths and weaknesses of the different methods and the potential benefit from their combined implementation. After study approval by the local Ethics Committee, 47 patients (31 male, 16 female, age range: 24-88 years) were recruited from the Department of Dermatology of the University Hospital Jena. In this work, we present an illustrative selection of eleven cases from a clinical study combining in vivo MPT with in vivo CLSM. The patients presented with a broad range of dermatological disorders including seborrheic keratoses, angioma, actinic keratoses, melanocytic nevi, malignant melanoma, psoriasis, pemphigus vulgaris and scarring. Both methods, CLSM and MPT, were found to be suitable for in vivo imaging of superficial skin layers and may therefore be useful in dermatological practice for the diagnosis of skin diseases. However, both methods differ in their technical and physical principles. Thus, despite of many similarities concerning the morphological presentation of cells and tissues, important differences are recognized. Synergies of the combination of CLSM and MPT may be obtained by combined implementation in order to benefit from the fast overview given by CLSM and the detailed imaging of skin structures by MPT.

 

The bulge area is the major hair follicle source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla

Liu F, Uchugonova A, Kimura H, Zhang C, Zhao M, Zhang L, Koenig K, Duong J, Aki R, Saito N, Mii S, Amoh Y, Katsuoka K, Hoffman RM.

Cell Cycle. 2011 Mar 1;10(5):830-9. Epub 2011 Mar 1.

Abstract:

Nestin has been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be pluripotent and be able to form neurons and other non-follicle cell types. The nestin-expressing pluripotent stem cells from the DP have been termed skin precursor or SKP cells. The objective of the present study was to determine the major source of nestin-expressing pluripotent stem cells in the hair follicle and to compare the ability of the nestin-expressing pluripotent stem cells from the BA and DP to repair spinal cord injury. Transgenic mice in which the nestin promoter drives GFP (ND-GFP) were used in order to observe nestin expression in the BA and DP. Nestin-expressing DP cells were found in early and middle anagen. The BA had nestin expression throughout the hair cycle and to a greater extent than the DP. The cells from both regions had very long processes extending from them as shown by two-photon confocal microscopy. Nestin-expressing stem cells from both areas differentiated into neuronal cells at high frequency in vitro. Both nestin-expressing DP and BA cells differentiated into neuronal and glial cells after transplantation to the injured spinal cord and enhanced injury repair and locomotor recovery within four weeks. Nestin-expressing pluripotent stem cells from both the BA and DP have potential for spinal cord regeneration, with the BA being the greater and more constant source.

 

The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle

Uchugonova A, Duong J, Zhang N, König K, Hoffman RM.

J Cell Biochem. 2011 Aug;112(8):2046-50. doi: 10.1002/jcb.23122.

Abstract:

Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestin-expressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestin-expressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestin-expressing dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 µm with long extrusions, as shown by 2-photon imaging. In the present study, using 2-photon imaging of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, and during wound healing, suggesting that the BA may be the source of the stem cells of the skin itself.

 

Watching stem cells in the skin of living mice noninvasively

Uchugonova A, Hoffman RM, Weinigel M, Koenig K.

Cell Cycle. 2011 Jun 15;10(12):2017-20. Epub 2011 Jun 15.

Abstract:

We demonstrate noninvasive, high-resolution multiphoton tomography of nestin-expressing stem cells of hair follicles in living transgenic nude mice. An imaging system comprised of a compact femtosecond laser, 3D scan head mounted on a flexible mechano-optical articulated arm for simultaneous intra-tissue fluorescence and second harmonic detection (SHG) detection was used. This noninvasive method enables long-term in vivo tracking of intra-tissue stem cells in living animals. Multiphoton animal sectioning with subcellular resolution can visualize the real-time behavior of single stem cells in their native tissue microenvironment.

 

Keratinocyte morphology of human skin evaluated by in vivo multiphoton laser tomography

Koehler MJ, Zimmermann S, Springer S, Elsner P, König K, Kaatz M.

Skin Res Technol. 2011 Nov;17(4):479-86. doi: 10.1111/j.1600-0846.2011.00522.x. Epub 2011 Mar 31.

Abstract:

BACKGROUND:  Multiphoton tomography (MPT) is a novel non-invasive imaging method in dermatology allowing the depiction of the epidermis with sub-cellular resolution. Here, we present a descriptive characterization of unaffected human epidermis, morphometric data on human keratinocytes and some epidermal parameters in vivo and a morphological characterization of keratinocyte changes in actinic keratoses. METHODS:  In a clinical setting, 57 volunteers of different age groups were examined using MPT. RESULTS:  The morphological appearance of keratinocytes showed polygonal cells in the horny layer, a granular cytoplasm in the stratum granulosum, smaller prickle cells in the stratum spinosum and hyperpigmented small round basal cells. Actinic keratoses presented remarkable differences including widened inter-cellular spaces, heterogeneity in cellular fluorescence and shape as well as an increased ratio of nuclear to cellular size. Finally, the thickness of the epidermis was significantly increased in actinic keratoses compared with the control. CONCLUSION:  In vivo MPT provides high-resolution images allowing the identification and quantification of cellular morphometric parameters. First observations of morphology and morphometry of actinic keratoses are reported.

 

Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy

Studier H, Breunig HG, König K.

J Biophotonics. 2011 Jan;4(1-2):84-91. doi: 10.1002/jbio.201000010. Epub 2010 Mar 10.

Abstract:

Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti: sapphire oscillator that produced 100 fs pulses (spectral width ∼10 nm) and two ultrabroadband Ti: sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen.