High accuracy 4D cell tracking into explanted skin using two-photon excitation microscopy.
El Mourdi I,Canivet A,Joncker N T,Bellard E,Allart S
Microscopy research and technique
Two-photon excitation microscopy (2PEM) analysis of large explanted organs is still laborious, principally because of tissue movements inducing lateral and axial drifts during extended imaging sessions. Here, we describe a two-step approach to track motile T cells in murine dorsal explanted skin with the best accuracy. First, we compared various explanted skin mounting methods for 2PEM analysis to define the setup allowing for minimal sample drift over time. Second, we developed two algorithms with the ImageJ software (National Institute of Health, Bethesda, MD) to correct the residual drift using lateral and axial registration of the collagen network. Finally, we applied the macro we developed to track fluorescent T cells in explanted skin. We found that our newly developed macro is more efficient than freely or commercially available software for shift correction, leading to more accurate velocity calculations. Our work provides a practical guide for investigators interested to employ skin-imaging approaches and offers a free alternative to commercial software for correcting lateral and axial drifts.
10.1002/jemt.22474
In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy.
Jang Won Hyuk,Shim Sehwan,Wang Taejun,Yoon Yeoreum,Jang Won-Suk,Myung Jae Kyung,Park Sunhoo,Kim Ki Hean
Scientific reports
Ionizing radiation (IR) injury is tissue damage caused by high energy electromagnetic waves such as X-ray and gamma ray. Diagnosis and treatment of IR injury are difficult due to its characteristics of clinically latent post-irradiation periods and the following successive and unpredictable inflammatory bursts. Skin is one of the many sensitive organs to IR and bears local injury upon exposure. Early-stage diagnosis of IR skin injury is essential in order to maximize treatment efficiency and to prevent the aggravation of IR injury. In this study, early-stage changes of the IR injured skin at the cellular level were characterized in an in vivo mouse model by two-photon microscopy (TPM). Various IR doses were applied to the mouse hind limbs and the injured skin regions were imaged daily for 6 days after IR irradiation. Changes in the morphology and distribution of the epidermal cells and damage of the sebaceous glands were observed before clinical symptoms. These results showed that TPM is sensitive to early-stage changes of IR skin injury and may be useful for its diagnosis.
10.1038/srep19216
Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra.
Laiho Lily H,Pelet Serge,Hancewicz Thomas M,Kaplan Peter D,So Peter T C
Journal of biomedical optics
Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.
10.1117/1.1891370
Microfabrication of polymer microneedle arrays using two-photon polymerization.
Mckee Seyyedhossien,Lutey Adrian,Sciancalepore Corrado,Poli Federica,Selleri Stefano,Cucinotta Annamaria
Journal of photochemistry and photobiology. B, Biology
Three dimensional (3D) printing technology has pushed state-of-the-art manufacturing towards more advanced processing methods through its ability to produce complex computer-designed 3D structures in a wide range of materials. Two-photon polymerization applied to the fabrication of ultraprecise 3D microstructures is one of the various innovative approaches to cutting-edge 3D printing. The integration of an ultrashort pulsed laser source and an appropriate photoresist has made it an attractive candidate for advanced photonics and biomedical applications. This paper presents the development of 3D solid microneedle arrays as a novel transdermal drug delivery system via two-photon polymerization in a single manufacturing step. Through a series of experiments, the best fabrication parameters are identified. Finite element simulations are then performed to investigate the interaction between a single microneedle and human skin. The results of this study highlight the influence of fabrication parameters such as laser power, scanning speed, hatch distance and layer height on the structural resolution and fabrication time of microneedles, as well as human skin deformation caused through application of force to a single polymer microneedle.
10.1016/j.jphotobiol.2022.112424
Two Photon Intravital Microscopy of Lyme Borrelia in Mice.
Belperron Alexia A,Mao Jialing,Bockenstedt Linda K
Methods in molecular biology (Clifton, N.J.)
Two-photon intravital microscopy is a powerful tool that allows visualization of cells in intact tissues in a live animal in real time. In recent years, this advanced technology has been applied to understand pathogen-host interactions using fluorescently labeled bacteria. In particular, infectious fluorescent transformants of the Lyme disease spirochete Borrelia burgdorferi, an Ixodes tick-transmitted pathogen, have been imaged by two-photon intravital microscopy to study bacterial motility and interactions of the pathogen with feeding ticks and host tissues. Here, we describe the techniques and equipment used to image mammalian-adapted spirochetes in the skin of living mice in vivo and in joints ex vivo using two-photon intravital microscopy.
10.1007/978-1-4939-7383-5_20
Imaging directed photothermolysis through two-photon absorption demonstrated on mouse skin - a potential novel tool for highly targeted skin treatment.
Wang Hequn,Zandi Soodabeh,Lee Anthony M D,Zhao Jianhua,Lui Harvey,McLean David I,Zeng Haishan
Journal of biophotonics
One-photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un-intended tissue damage. Our objective is to test whether two-photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second-harmonic-generation (SHG) and two-photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non-invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA-based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two-photon absorption photothermolysis method could potentially be applied in clinical dermatology.
10.1002/jbio.201300016
Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy.
Bentolila Nathan Y,Barnhill Raymond L,Lugassy Claire,Bentolila Laurent A
Methods in molecular biology (Clifton, N.J.)
Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.
10.1007/978-1-4939-7724-6_15
Quantification and visualization of cellular NAD(P)H in young and aged female facial skin with in vivo two-photon tomography.
Miyamoto K,Kudoh H
The British journal of dermatology
BACKGROUND:In vivo two-photon tomography is a novel noninvasive three-dimensional optical skin imaging technology with subcellular resolution which enables the sensitive detection of endogenous fluorophores. One of these fluorophores, NAD(P)H (a coenzyme which plays an important role in the release of free energy during glycolysis, and influences filaggrin and lipid synthesis), can be selectively detected in keratinocytes (granular cells) with two-photon tomography. OBJECTIVES:To quantify NAD(P)H levels in subsurface human facial skin in vivo as a measure to determine if there are changes with age. METHODS:A total of 80 healthy Asian females were enrolled in this study, aged 21-68 years. Measurements were performed on facial skin using in vivo two-photon tomography (DermaInspect/MPTflex™, JenLab GmbH, Jena, Germany). The laser beam scans a skin field of interest in pulses, focused at a depth to reach the granular layer. The near-infrared laser pulses excite the endogenous fluorophores NAD(P)H. Image processing was performed to obtain high-resolution autofluorescence images (optical biopsies) and to quantify the fluorescent grey scale to determine NAD(P)H levels. Additional skin surface measures taken were hydration (corneometer), elasticity (cutometer) and wrinkles (image capture and analysis). RESULTS:Statistically significant changes in all measured parameters as a function of age were observed. Most importantly, the mean fluorescent grey scale values for NAD(P)H in the youngest group studied (women in their 20s) was 38.8 (SD ± 12.39), while that of the oldest group studied (women in their 60s) was 32.7 (SD ± 12.47). These NAD(P)H levels are statistically significantly different (P = 0.0078). CONCLUSIONS:The level of NAD(P)H in the epidermis is significantly greater in younger vs. older skin in vivo. This likely reflects decreased production and/or increased degradation of NAD(P)H in older skin, possibly as a result of chronological ageing and environmental damage (e.g. photodamage). NAD(P)H levels in epidermal skin may be a useful biomarker of skin ageing in vivo. It is also likely that maintaining NAD(P)H production is a useful approach to maintaining good skin condition and caring for ageing skin.
10.1111/bjd.12370
Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.
Hanson Kerry M,Behne Martin J,Barry Nicholas P,Mauro Theodora M,Gratton Enrico,Clegg Robert M
Biophysical journal
Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.
10.1016/S0006-3495(02)73936-2
Propionate alleviates itch in murine models of atopic dermatitis by modulating sensory TRP channels of dorsal root ganglion.
Allergy
BACKGROUND:Itch is the most common symptom of atopic dermatitis (AD) and significantly decreases the quality of life. Skin microbiome is involved in AD pathogenesis, whereas its role in the regulation of itch remains elusive. In this study, we aimed to investigate the effects of skin microbial metabolite propionate on acute and chronic pruritus and to explore the mechanism. METHODS:Using various mouse models of itch, the roles of propionate were explored by behavioral tests and histopathology/immunofluorescent analysis. Primary-cultured dorsal root ganglion neurons and HEK293 cells expressing recombinant human TRP channels were utilized for in vitro calcium imaging/in vivo miniature two-photon imaging in combination with electrophysiology and molecular docking approaches for investigation of the mechanism. RESULTS:Propionate significantly alleviated itch and alloknesis in various mouse models of pruritus and AD and decreased the density of intraepidermal nerve fibers. Propionate reduced the responsiveness of dorsal root ganglion neurons to pruritogens in vitro, attenuated the hyper-excitability in sensory neurons in MC903-induced AD model, and inhibited capsaicin-evoked hTRPV1 currents (IC = 20.08 ± 1.11 μM) via interacting with the vanilloid binding site. Propionate also decreased the secretion of calcitonin gene-related peptide by nerves in MC903-induced AD mouse model, which further attenuated itch and skin inflammation. CONCLUSION:Our study revealed a protective effect of propionate against persistent itch through direct modulation of sensory TRP channels and neuropeptide production in neurons. Regulation of itch via the skin microbiome might be a novel strategy for the treatment of AD.
10.1111/all.15998
Clinical two-photon microendoscopy.
König K,Ehlers A,Riemann I,Schenkl S,Bückle R,Kaatz M
Microscopy research and technique
Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope. The microendoscope was attached to the multiphoton tomograph DermaInspect and employed to detect the extracellular matrix proteins collagen and elastin in the human dermis of volunteers and patients with ulcera by in vivo second harmonic generation and in vivo two-photon autofluorescence.
10.1002/jemt.20445
Investigating the race for the surface and skin integration in clinically retrieved abutments with two-photon microscopy.
Kapsokalyvas D,van Hoof M,Wigren S,Chimhanda T,Kuijpers H J,Ramaekers F C S,Stokroos R J,van Zandvoort M A M J
Colloids and surfaces. B, Biointerfaces
Bone conduction hearing implants can rehabilitate some types of hearing loss. A hydroxyapatite (HA)-coated skin-penetrating abutment was developed to allow for soft tissue preservation and increased skin-abutment adherence. Inflammation is thought to relate to bacterial infection of pockets around the abutment. Upon integration, the host's ability to cover the abutment surface ("race for the surface"), and thus control and prevent competitive bacteria from colonizing it, is improved. However, the attachment mechanisms behind it are not clear. In this study, we applied two-photon microscopy to visualize tissue attachment on abutments retrieved from patients. Skin integration markers were validated and applied to four HA-coated abutments. Evidence of skin integration was found, including the presence of hemidesmosomes, a basement membrane, dermal collagen and vascularization. Cases with clinical signs of severe inflammation and evident biofilm formation showed limited skin integration based on these indicators, confirming the applicability of the "race for the surface" model.
10.1016/j.colsurfb.2017.07.072
Two-photon microscopy of deep intravital tissues and its merits in clinical research.
Wang B-G,König K,Halbhuber K-J
Journal of microscopy
Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.
10.1111/j.1365-2818.2009.03330.x
Penetration Depth of Propylene Glycol, Sodium Fluorescein and Nile Red into the Skin Using Non-Invasive Two-Photon Excited FLIM.
Pharmaceutics
The stratum corneum () forms a strong barrier against topical drug delivery. Therefore, understanding the penetration depth and pathways into the is important for the efficiency of drug delivery and cosmetic safety. In this study, TPT-FLIM (two-photon tomography combined with fluorescence lifetime imaging) was applied as a non-invasive optical method for the visualization of skin structure and components to study penetration depths of exemplary substances, like hydrophilic propylene glycol (), sodium fluorescein () and lipophilic Nile red () into porcine ear skin ex vivo. Non-fluorescent was detected indirectly based on the pH-dependent increase in the fluorescence lifetime of components. The pH similarity between and viable epidermis limited the detection of . reached the viable epidermis, which was also proved by laser scanning microscopy. Tape stripping and confocal Raman micro-spectroscopy were performed additionally to study , which revealed penetration depths of ≈5 and ≈8 μm, respectively. Lastly, did not permeate the . We concluded that the amplitude-weighted mean fluorescence lifetime is the most appropriate FLIM parameter to build up penetration profiles. This work is anticipated to provide a non-invasive TPT-FLIM method for studying the penetration of topically applied drugs and cosmetics into the skin.
10.3390/pharmaceutics14091790
Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging.
Lou Xiaoding,Zhao Zujin,Tang Ben Zhong
Small (Weinheim an der Bergstrasse, Germany)
Two-photon fluorescence imaging technique is a powerful bioanalytical approach in terms of high photostability, low photodamage, high spatiotemporal resolution. Recently, fluorescent organic dots comprised of organic emissive cores and a polymeric matrix are emerging as promising contrast reagents for two-photon fluorescence imaging, owing to their numerous merits of high and tunable fluorescence, good biocompatibility, strong photobleaching resistance, and multiple surface functionality. The emissive core is crucial for organic dots to get high brightness but many conventional chromophores often encounter a severe problem of fluorescence quenching when they form aggregates. To solve this problem, fluorogens featuring aggregation-induced emission (AIE) can fluoresce strongly in aggregates, and thus become ideal candidates for fluorescent organic dots. In addition, two-photon absorption property of the dots can be readily improved by just increase loading contents of AIE fluorogen (AIEgen). Hence, organic dots based on AIEgens have exhibited excellent performances in two-photon fluorescence in vitro cellular imaging, and in vivo vascular architecture visualization of mouse skin, muscle, brain and skull bone. In view of the rapid advances in this important research field, here, we highlight representative fluorescent organic dots with an emissive core of AIEgen aggregate, and discuss their great potential in bioimaging applications.
10.1002/smll.201600872
In vivo quantitative molecular absorption of glycerol in human skin using coherent anti-Stokes Raman scattering (CARS) and two-photon auto-fluorescence.
Sarri Barbara,Chen Xueqin,Canonge Rafaël,Grégoire Sébastien,Formanek Florian,Galey Jean-Baptiste,Potter Anne,Bornschlögl Thomas,Rigneault Hervé
Journal of controlled release : official journal of the Controlled Release Society
The penetration of small molecules through the human skin is a major issue for both safety and efficacy issues in cosmetics and pharmaceutic domains. To date, the quantification of active molecular compounds in human skin following a topical application uses ex vivo skin samples mounted on Franz cell diffusion set-up together with appropriate analytical methods. Coherent anti-Stokes Raman scattering (CARS) has also been used to perform active molecule quantification on ex vivo skin samples, but no quantification has been described in human skin in vivo. Here we introduce and validate a framework for imaging and quantifying the active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework was exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time was demonstrated, showing that, contrary to water, the xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time. More generally, the proposed imaging framework provides an enabling platform for establishing functional activity of topically applied products in vivo.
10.1016/j.jconrel.2019.07.018
Two-photon laser-scanning fluorescence microscopy applied for studies of human skin.
Ericson M B,Simonsson C,Guldbrand S,Ljungblad C,Paoli J,Smedh M
Journal of biophotonics
Two-photon laser scanning fluorescence microscopy (TPM) has been shown to be advantageous for imaging optically turbid media such as human skin. The ability of performing three-dimensional imaging without presectioning of the samples makes the technique not only suitable for noninvasive diagnostics but also for studies of topical delivery of xenobiotics. Here, TPM is used as a method to visualize both autofluorescent and exogenous fluorophores in skin. Samples exposed to sulforhodamine B have been scanned from two directions to investigate attenuation effects. It is shown that optical effects play a major role. Thus, TPM is excellent for visualizing the localization and distribution of fluorophores in human skin, although quantification might be difficult. Furthermore, an image-analysis algorithm has been implemented to facilitate interpretation of TPM images of autofluorescent features of nonmelanoma skin cancer obtained ex vivo. The algorithm was designed to detect cell nuclei and currently has a sensitivity and specificity of 82% and 78% to single cell nuclei. However, in order to detect multinucleated cells, the algorithm needs further development.
10.1002/jbio.200810022
Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores.
Biomedical optics express
The noninvasive two-photon excitation autofluorescence imaging of cellular and subcellular structure and dynamics in live tissue could provide critical information for biomedical studies. However, the two-photon microscopy of short-wavelength endogenous fluorophores, such as tryptophan and hemoglobin, is extremely limited due to the lack of suitable imaging techniques. In this study, we developed a short-wavelength excitation time- and spectrum-resolved two-photon microscopy system. A 520-nm femtosecond fiber laser was used as the excitation source, and a time-correlated single-photon counting module connected with a spectrograph was used to provide time- and spectrum-resolved detection capability. The system was specially designed for measuring ultraviolet and violet-blue fluorescence signals and thus was very suitable for imaging short-wavelength endogenous fluorophores. Using the system, we systematically compared the fluorescence spectra and fluorescence lifetimes of short-wavelength endogenous fluorophores, including the fluorescent molecules tyrosine, tryptophan, serotonin (5-HT), niacin (vitamin B3), pyridoxine (vitamin B6), and NADH and the protein group (keratin, elastin, and hemoglobin). Then, high-resolution three-dimensional (3D) label-free imaging of different biological tissues, including rat esophageal tissue, rat oral cheek tissue, and mouse ear skin, was performed or . Finally, we conducted time-lapse imaging of leukocyte migration in the lipopolysaccharide injection immunization model and a mechanical trauma immunization model. The results indicate that the system can specifically characterize short-wavelength endogenous fluorophores and provide noninvasive label-free 3D visualization of fine structures and dynamics in biological systems. The microscopy system developed here can empower more flexible imaging of endogenous fluorophores and provide a novel method for the 3D monitoring of biological events in their native environment.
10.1364/BOE.493015
Two-Photon-Excited Silica and Organosilica Nanoparticles for Spatiotemporal Cancer Treatment.
Croissant Jonas G,Zink Jeffrey I,Raehm Laurence,Durand Jean-Olivier
Advanced healthcare materials
Coherent two-photon-excited (TPE) therapy in the near-infrared (NIR) provides safer cancer treatments than current therapies lacking spatial and temporal selectivities because it is characterized by a 3D spatial resolution of 1 µm and very low scattering. In this review, the principle of TPE and its significance in combination with organosilica nanoparticles (NPs) are introduced and then studies involving the design of pioneering TPE-NIR organosilica nanomaterials are discussed for bioimaging, drug delivery, and photodynamic therapy. Organosilica nanoparticles and their rich and well-established chemistry, tunable composition, porosity, size, and morphology provide ideal platforms for minimal side-effect therapies via TPE-NIR. Mesoporous silica and organosilica nanoparticles endowed with high surface areas can be functionalized to carry hydrophobic and biologically unstable two-photon absorbers for drug delivery and diagnosis. Currently, most light-actuated clinical therapeutic applications with NPs involve photodynamic therapy by singlet oxygen generation, but low photosensitizing efficiencies, tumor resistance, and lack of spatial resolution limit their applicability. On the contrary, higher photosensitizing yields, versatile therapies, and a unique spatial resolution are available with engineered two-photon-sensitive organosilica particles that selectively impact tumors while healthy tissues remain untouched. Patients suffering pathologies such as retinoblastoma, breast, and skin cancers will greatly benefit from TPE-NIR ultrasensitive diagnosis and therapy.
10.1002/adhm.201701248
Two-photon microscopy of dermal innervation in a human re-innervated model of skin.
Sevrain David,Le Grand Yann,Buhé Virginie,Jeanmaire Christine,Pauly Gilles,Carré Jean-Luc,Misery Laurent,Lebonvallet Nicolas
Experimental dermatology
When skin is injured, innervation can be severely disrupted. The subsequent re-innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two-photon excitation fluorescence (TPEF) microscopy of entire human skin explants re-innervated by rodent sensory neurons labelled with the styryl dye FM1-43. TPEF imaging of nerve fibres to a depth up to roughly 300 μm within the dermis was demonstrated, allowing three-dimensional reconstruction of the neural tree structure. Endogenous second-harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.
10.1111/exd.12108
Photodamage in deep tissue two-photon optical biopsy of human skin.
Dalbosco Luca,Zanini Giulia,D'Amato Elvira,Tessarolo Francesco,Boi Sebastiana,Bauer Paolo,Haase Albrecht,Antolini Renzo
Journal of biophotonics
Photodamage, induced by femtosecond laser radiation, was studied in thick samples of human skin tissue (healthy skin and neoplastic lesions). Photobleaching, photoionization, and thermomechanical damage effects were characterized comparatively. The laser power dependence of the damage rates allowed to connect macroscopic effects to underlying molecular processes. Optical effects were correlated to histopathological changes. Tissue alterations were found only from thermomechanical cavitation and limited to superficial layers of the epidermis. From the depth-dependencies of all damage thresholds a depth-dependent power-compensation scheme was defined allowing for damage-free deep tissue optical biopsy. Damage-induced luminescence pattern for different excitation powers and a corresponding threshold analysis.
10.1002/jbio.201400083
Quantitative two-photon microscopy imaging analysis of human skin to evaluate enhanced transdermal delivery by hybrid-type multi-lamellar nanostructure.
Ahn Jinhyo,Kim Kyeong Hu,Choe Kibaek,Lim Joo Hyuck,Lee Seung Ki,Kim Yeon Sook,Kim Pilhan
Biomedical optics express
Transdermal skin delivery is a method to transport various topical formulations to a deeper skin layer non-invasively. Permeability analysis of many delivering agents has been mostly conducted by a simple tape stripping method. However, it cannot reveal a detailed depth-dependent distribution profile of transdermally delivered agents in the skin. In this work, we achieved a cellular-level depth-defined visualization of fluorophore-labelled human epidermal growth factor (EGF) transdermally delivered to human skin by using encapsulation with common liposomes and newly fabricated multi-lamellar nanostructures using a custom-design two-photon microscopy system. It was able to generate 3D reconstructed images displaying the distribution of human EGF inside the human skin sample with high-resolution. Based on a depthwise fluorescence intensity profile showing the permeation of human EGF, a quantitative analysis was performed to assess the transdermal delivery efficacy achieved by each formulation, showing a significant improvement of the efficacy with the utilization of multi-lamellar nanostructure.
10.1364/BOE.9.003974
Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues.
Yu Yingqiu,Lee Anthony M D,Wang Hequn,Tang Shuo,Zhao Jianhua,Lui Harvey,Zeng Haishan
Journal of biomedical optics
There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.
10.1117/1.JBO.17.7.077004
Translation of two-photon microscopy to the clinic: multimodal multiphoton CARS tomography of in vivo human skin.
Journal of biomedical optics
<p>Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for <italic>in vivo</italic> 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 <italic>in vivo</italic> 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 <italic>in vivo</italic> skin histology to realize rapid early diagnosis as well as treatment control.</p>.
10.1117/1.JBO.25.1.014515
Two-photon and second harmonic microscopy in clinical and translational cancer research.
Perry Seth W,Burke Ryan M,Brown Edward B
Annals of biomedical engineering
Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM-two-photon excitation fluorescence microscopy, and second harmonic generation microscopy-as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.
10.1007/s10439-012-0512-9
High-contrast visualization of human skin cancers with combined reflectance confocal and moxifloxacin-based two-photon microscopy: An ex vivo study.
Lasers in surgery and medicine
BACKGROUND AND OBJECTIVES:Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin-based two-photon (MB-TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods. MATERIALS AND METHODS:Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3-5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology. RESULTS:Combined RC and MB-TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination. CONCLUSIONS:The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.
10.1002/lsm.23600
Organotypic cell cultures and two-photon imaging: tools for in vitro and in vivo assessment of percutaneous drug delivery and skin toxicity.
Pappinen Sari,Pryazhnikov Evgeny,Khiroug Leonard,Ericson Marica B,Yliperttula Marjo,Urtti Arto
Journal of controlled release : official journal of the Controlled Release Society
The outermost protective layer of the skin, the stratum corneum, is responsible for skin impermeability toward external medications and potentially harmful chemicals. Stratum corneum is the target for physical and chemical approaches to enhance drug permeation. These approaches are commonly investigated in the field of drug delivery, but the drug absorption enhancement is often linked with local toxicity. In this review we are discussing two emerging technologies for drug and chemical studies in the skin: organotypic cell cultures and non-invasive two-photon microscopic imaging. Even though several cell culture based 'skin equivalents' have been introduced and validated for skin irritation testing, they are usually leaky and inadequately characterized in terms of permeation. Rat epidermal culture model (ROC) has been thoroughly characterized and it shows comparable barrier properties with the human skin thereby being useful in drug permeation and toxicity studies. In vitro and in vivo visualizations of permeants and skin structures are now feasible due to the rapid development of two-photon microscopy that allows improved depth scanning and direct in vivo visualization of the permeating compounds and adverse reactions in the skin structures. In summary, the new tools in percutaneous drug delivery studies will provide new insights to the permeation process and local toxicity. These tools may facilitate development of effective and safe transdermal drug delivery methods.
10.1016/j.jconrel.2012.03.005
Two-photon microscopy for intracutaneous imaging of stem cell activity in mice.
Experimental dermatology
The adult skin is a typical example of a highly regenerative tissue. Terminally differentiated keratinocytes are shed from the external layers of the epidermis or extruded from the skin as part of the growing hair shaft on a daily basis. These are effectively replenished through the activity of skin-resident stem cells. Precise regulation of stem cell activity is critical for normal skin homoeostasis or wound healing and irregular stem cell proliferation or differentiation can lead to skin disease. The scarcity and dynamic nature of stem cells presents a major challenge for elucidating their mechanism of action. To address this, we have recently established a system for visualizing stem cell activity, in real time or long term, in the intact skin of live mice using two-photon microscopy. The purpose of this review was to provide essential information to researchers who wish to incorporate two-photon microscopy and live imaging into their experimental toolbox for studying aspects of skin and stem biology in the mouse model. We discuss fundamental principles of the method, instrumentation and basic experimental approaches to interrogate stem cell activity in the interfollicular epidermis and hair follicle.
10.1111/exd.13221
Non-Invasive Skin Imaging Assessment of Human Stress During Head-Down Bed Rest Using a Portable Handheld Two-Photon Microscope.
Frontiers in physiology
Spaceflight presents a series of physiological and pathological challenges to astronauts resulting from ionizing radiation, microgravity, isolation, and other spaceflight hazards. These risks cause a series of aging-related diseases associated with increased oxidative stress and mitochondria dysfunction. The skin contains many autofluorescent substances, such as nicotinamide adenine dinucleotide phosphate (NAD(P)H), keratin, melanin, elastin, and collagen, which reflect physiological and pathological changes . In this study, we used a portable handheld two-photon microscope to conduct high-resolution skin imaging on volunteers during 15 days of head-down bed rest. The two-photon microscope, equipped with a flexible handheld scanning head, was used to measure two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of the left forearm, left front chest, and forehead of volunteers. Changes in TPEF, SHG, and the extended SHG-to-AF(TPEF) aging index of the dermis (SAAID) were measured. It was found that TPEF intensity increased during bed rest and was restored to normal levels after recovery. Meanwhile, SHG increased slightly during bed rest, and the skin aging index increased. Moreover, we found the skin TPEF signals of the left forearm were significantly negatively associated with the oxidative stress marker malondialdehyde (MDA) and DNA damage marker 8-hydroxy-2'-desoxyguanosine (8-OHdG) values of subjects during head-down bed rest. Meanwhile, the SHG signals were also significantly negatively correlated with MDA and 8-OHDG. A significant negative correlation between the extended SAAID of the left chest and serum antioxidant superoxide dismutase (SOD) levels was also found. These results demonstrate that skin autofluorescence signals can reflect changes in human oxidant status. This study provides evidence for in-orbit monitoring of changes in human stress using a portable handheld two-photon microscope for skin imaging.
10.3389/fphys.2022.899830
An overview of microneedle applications, materials, and fabrication methods.
Beilstein journal of nanotechnology
Microneedle-based microdevices promise to expand the scope for delivery of vaccines and therapeutic agents through the skin and withdrawing biofluids for point-of-care diagnostics - so-called theranostics. Unskilled and painless applications of microneedle patches for blood collection or drug delivery are two of the advantages of microneedle arrays over hypodermic needles. Developing the necessary microneedle fabrication processes has the potential to dramatically impact the health care delivery system by changing the landscape of fluid sampling and subcutaneous drug delivery. Microneedle designs which range from sub-micron to millimetre feature sizes are fabricated using the tools of the microelectronics industry from metals, silicon, and polymers. Various types of subtractive and additive manufacturing processes have been used to manufacture microneedles, but the development of microneedle-based systems using conventional subtractive methods has been constrained by the limitations and high cost of microfabrication technology. Additive manufacturing processes such as 3D printing and two-photon polymerization fabrication are promising transformative technologies developed in recent years. The present article provides an overview of microneedle systems applications, designs, material selection, and manufacturing methods.
10.3762/bjnano.12.77
Skin Imaging Using Ultrasound Imaging, Optical Coherence Tomography, Confocal Microscopy, and Two-Photon Microscopy in Cutaneous Oncology.
Oh Byung Ho,Kim Ki Hean,Chung Kee Yang
Frontiers in medicine
With the recognition of dermoscopy as a new medical technology and its available fee assessment in Korea comes an increased interest in imaging-based dermatological diagnosis. For the dermatologist, who treats benign tumors and malignant skin cancers, imaging-based evaluations can assist with determining the surgical method and future follow-up plans. The identification of the tumor's location and the existence of blood vessels can guide safe treatment and enable the use of minimal incisions. The recent development of high-resolution microscopy based on laser reflection has enabled observation of the skin at the cellular level. Despite the limitation of a shallow imaging depth, non-invasive light-based histopathologic examinations are being investigated as a rapid and pain-free process that would be appreciated by patients and feature reduced time from consultation to treatment. In the United States, the current procedural terminology billing code was established for reflectance confocal microscopy in 2016 and has been used for the skin cancer diagnosis ever since. In this review, we introduce the basic concepts and images of ultrasound imaging, optical coherence tomography, confocal microscopy, and two-photon microscopy and discuss how they can be utilized in the field of dermatological oncology.
10.3389/fmed.2019.00274
Use of two-photon microscopy to study Leishmania major infection of the skin.
Carneiro Matheus Batista,Hohman Leah Shan,Egen Jackson G,Peters Nathan C
Methods (San Diego, Calif.)
Intra-vital two-photon microscopy (2P-IVM) allows for in-situ investigation of tissue organization, cell behavior and the dynamic interactions between different cell types in their natural environment. This methodology has also expanded our understanding of the immune response against pathogens. Leishmania are protozoan intracellular parasites that have adapted to successfully establish infection within the context of an inflammatory response in the skin following transmission by the bite of an infected sand fly. The generation of fluorescent transgenic parasites coupled with the increased availability of different types of fluorescent transgenic reporter mice has facilitated the study of the host-parasite interaction in the skin, significantly impacting our understanding of cutaneous leishmaniasis. In this review we will discuss 2P-IVM in the context of Leishmania infection of the mouse ear skin and describe a simple and minimally invasive procedure that allows long-term imaging of this host-pathogen interaction.
10.1016/j.ymeth.2017.04.012
Research Techniques Made Simple: Two-Photon Intravital Imaging of the Skin.
Obeidy Peyman,Tong Philip L,Weninger Wolfgang
The Journal of investigative dermatology
Over the last few years, intravital two-photon microscopy has matured into a powerful technology helping basic and clinical researchers obtain quantifiable details of complex biological mechanisms in live and intact tissues. Two-photon microscopy provides high spatial and temporal resolution in vivo with little phototoxicity that is unattainable by other optical tools like confocal microscopy. Using ultrashort laser pulses, two-photon microscopy allows the visualization of molecules, cells, and extracellular structures up to depths of 1 mm within tissues. Consequently, real-time imaging of the individual skin layers under both physiological and pathological conditions has revolutionized our understanding of cutaneous homeostasis, immunity, and tumor biology. This review provides an overview to two-photon microscopy of the skin by covering the basic concepts and current applications in diverse preclinical and clinical settings.
10.1016/j.jid.2018.01.017