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Topicals and chemical peels are the standard of care for management of facial hyperpigmentation. However, traditional therapies have come under recent scrutiny, such as topical hydroquinone (HQ) has some regulatory restrictions, and high concentration trichloroacetic acid (TCA) peel pose a risk in patients with skin of colour. The objective of our research was to identify, investigate and elucidate the mechanism of action of a novel TCA‐ and HQ‐free professional‐use chemical peel to manage common types of facial hyperpigmentation. Using computational modelling and in vitro assays on tyrosinase, we identified proprietary multi‐acid synergistic technology (MAST). After a single application on human skin explants, MAST peel was found to be more effective than a commercial HQ peel in inhibiting melanin (histochemical imaging and gene expression). All participants completed the case study (N = 9) without any adverse events. After administration of the MAST peel by a dermatologist, the scoring and VISIA photography reported improvements in hyperpigmentation, texture and erythema, which could be linked to underlying pathophysiological changes in skin after peeling, visualized by non‐invasive optical biopsy of face. Using reflectance confocal microscopy (VivaScope®) and multiphoton tomography (MPTflex™), we observed reduction in melanin, increase in metabolic activity of keratinocytes, and no signs of inflammatory cells after peeling. Subsequent swabbing of the cheek skin found no microbiota dysbiosis resulting from the chemical peel. The strong efficacy with minimum downtime and no adverse events could be linked to the synergistic action of the ingredients in the novel HQ‐ and TCA‐free professional peel technology. [ABSTRACT FROM AUTHOR]

The dissociative ionization of deuterium chloride (DCl) is investigated by employing femtosecond pulses at 805 nm for a range of laser powers as a function of the linear chirp of the laser pulses. For all laser powers a significant influence of the sign of the chirp on the product branching ratio (D.sup.+)(Cl.super.+) is observed.

For the first time, the time-resolved two-photon excited autophosphorescence of non-labeled biological specimens was investigated by phosphoresce lifetime imaging with microscopic spatial resolution. A modified multiphoton tomograph was employed to record both photoluminescence contributions, autofluorescence and autophosphorescence, simultaneously, induced by two-photon excitation using an 80 MHz near infrared femtosecond-pulse-laser scanning beam, an acousto-optic modulator, and a time-correlated single-photon counting module for lifetime measurements from the picosecond to the microsecond range. In particular, the two-photon-excited luminescence of thermally altered bones was imaged. A strong dependence of the phosphorescence intensity on exposure temperature, with a maximum emission for an exposure temperature of approximately 600 °C was observed. Furthermore, the phosphorescence lifetime data indicated a bi-exponential signal decay with both a faster few µs decay time in the range of 3–10 µs and a slower one in the range of 30–60 µs. The recording of fluorescence and phosphorescence allowed deriving the relative signal proportion as an unbiased measure of the temperature dependence. The measurements on thermally altered bones are of particular interest for application to forensic and archeological investigations. [ABSTRACT FROM AUTHOR]

The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. However, visualization of aggregated erythrocytes, which would settle the question, has always been challenging. Direct methods usually study erythrocytes in 2D situations or low hematocrit (∼1%). Indirect methods, such as scattering or electric measurements, provide insight on the suspension evolution, but cannot directly discriminate between open or percolating structures. Here, we achieved a direct probing of the structures formed by erythrocytes in blood at stasis. We focused on blood samples at rest with controlled hematocrit of 45%, from healthy donors, and report observations from three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. The three techniques, used in geometries with thickness from 150 μm to 3 mm, highlight that erythrocytes form a continuous network with characteristic cracks, i.e., a colloidal gel. The characteristic distance between the main cracks is of the order of ∼100 μm. A complete description of the structure then requires a field of view of the order of ∼1 mm, in order to obtain a statistically relevant number of structural elements. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups. [ABSTRACT FROM AUTHOR]

Abstract: Nothing represents our age better than our most outer layer: the skin. But the age of the skin may differ from the person's chronological age due to a complex interplay of external and internal factors. A non‐invasive method to determine the skin age on a microscopic level in vivo is clinical multiphoton tomography which allows for direct non‐invasive imaging of biological tissue with subcellular resolution. By directly detecting signals from the major structural proteins of the skin, elastin and collagen, a skin‐age parameter, the so‐called SAAID, can be derived. This elastin‐collagen‐based skin age is exclusively provided by multiphoton tomography. The parameter allows assessing the dermatological skin status and evaluating the effects of cosmetic products like anti‐ageing creams or physical procedures such as laser treatments on a microscopic scale in vivo. Furthermore, the skin‐age parameter provides information on the influence of UV exposure and other skin‐damaging processes and helps NASA and ESA to understand skin‐ageing effects and wound healing related problems due to extended stays in space. [ABSTRACT FROM AUTHOR]

The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cell's metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cell's morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cell's metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases. [ABSTRACT FROM AUTHOR]

The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius . After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime τ m (tau-m) at 0.4 ns and 1.0–1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. [ABSTRACT FROM AUTHOR]

We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user's practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation. [ABSTRACT FROM AUTHOR]

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD (P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions. [ABSTRACT FROM AUTHOR]

Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible. [ABSTRACT FROM AUTHOR]

We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided. [ABSTRACT FROM AUTHOR]

Reports on the toxicity of inorganic nanoparticles used in sunscreen, in particular of zinc oxide and titanium dioxide, have raised concerns about a possible particle penetration through the skin barrier. We used two-photon imaging to visualize the distribution of zinc-oxide nanoparticles after topical application of a commercially available sunscreen on human skin in vivo and to investigate a possible penetration of nanoparticles beyond the stratum corneum. Two-photon imaging is in particular suitable for these investigations since the excitation and the nonlinear signal light from zinc-oxide nanoparticles as well as the endogenous skin autofluorescence are all spectrally well-separated which allows a clear identification of the signal origin by detection in two-spectral channels. Furthermore, microscopic modifications in the cutaneous structure like skin wrinkles which exhibit different thicknesses of the dermal layers and at the same time are regions where nanoparticle accumulation can be specifically investigated. The results indicate no penetration of nanoparticle through the barrier of the stratum corneum of healthy skin even in microscopic wrinkles. [ABSTRACT FROM AUTHOR]

ABSTRACT We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in lab-on-a-chip devices. Microsc. Res. Tech. 77:974-979, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Thermally-induced changes in Arabidopsis thaliana leaves were investigated with a novel cryo microscope by multiphoton, fluorescence lifetime and spectral imaging as well as micro spectroscopy. Samples were excited with fs pulses in the near-infrared range and cooled/heated in a cryogenic chamber. The results show morphological changes in the chloroplast distribution as well as a shift from chlorophyll to cell-wall fluorescence with decreasing temperature. At temperatures below -40 °C, also second harmonic generation was observed. The measurements illustrate the suitability of multiphoton imaging to investigate thermally-induced changes at temperatures used for cryopreservation as well as for basic investigations of thermal effects on plant tissue in general (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin invivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level. Microsc. Res. Tech., 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for in vivo histology. We report on clinical multiphoton coherent anti-Stokes Raman spectroscopy (CARS) tomography based on two tunable ultrashort near-infrared laser beams for label-free in vivo multimodal skin imaging. The multiphoton biopsies were obtained with the compact tomograph "MPTflex-CARS" using a photonic crystal fiber, an optomechanical articulated arm, and a four-detector-360 deg measurement head. The multiphoton tomograph has been employed to patients in a hospital with diseased skin. The clinical study involved 16 subjects, 8 patients with atopic dermatitis, 4 patients with psoriasis vulgaris, and 4 volunteers served as control. Two-photon cellular autofluorescence lifetime, second harmonic generation (SHG) of collagen, and CARS of intratissue lipids/proteins have been detected with single-photon sensitivity, submicron spatial resolution, and picosecond temporal resolution. The most important signal was the autofluorescence from nicotinamide adenine dinucleotide [NAD(P)H]. The SHG signal from collagen was mainly used to detect the epidermal–dermal junction and to calculate the ratio elastin/collagen. The CARS/Raman signal provided add-on information. Based on this view on the disease-affected skin on a subcellular level, skin areas affected by dermatitis and by psoriasis could be clearly identified. Multimodal multiphoton tomographs may become important label-free clinical high-resolution imaging tools for in vivo skin histology to realize rapid early diagnosis as well as treatment control. [ABSTRACT FROM AUTHOR]

Highlights: [•] A multiphoton cryomicroscope based on a low-power femtosecond fiber laser was built. [•] Multiphoton sectioning can be performed within ice. [•] Autofluorescence intensity and fluorescence lifetime increase during freezing. [•] Label-free optical biopsies of cryopreserved biomaterial can be obtained. [Copyright &y& Elsevier]

Purpose: The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation., Methods: Human corneas were imaged after different storage times: short-term (STS), medium-term (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition., Results: Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a1/a2) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a1/a2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a1/a2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 ± 190 cells/mm2 was obtained for MTS samples based on TPI., Conclusions: TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54-1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26-2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype., (© 2015 The Authors.)

In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting.