Multi-photon excited intensity and lifetime fluorescence images relying on endogenous contrast can be analyzed to quantify contributions from key metabolic co-enzymes and associated metabolic function and mitochondrial organization metrics. The high spatio-temporal resolution and context of these non-destructive measurements can be used to provide important insights related to a wide range of samples, conditions and disease models. Corresponding images are acquired from mitochondria, engineered tissues, excised and in vivo human tissues. Recent studies highlight the value of multi-parametric, label-free, metabolic assessments to improve our understanding of traumatic brain injury, (pre)cancer development, and vitiligo lesions.
Wound re-epithelialization is complex and imperfect. Understanding which cells are involved in this process, how they are spatially arranged, and when they contribute to wound healing is a longstanding scientific and clinical challenge. We used a recently developed fast large area multiphoton exoscope for in vivo imaging of human skin to study the process of wound healing in vivo in human skin. We monitored the re-epithelization of wounds generated by removal of the epidermis following a suction blistering procedure and identified the morphological and metabolic signatures of epidermal and dermal cells involved in the healing process.
Multiphoton microscopy (MPM) is a technology that can generate real-time, depth-resolved subsurface images of skin with histologic resolution and sensitivity based on endogenous molecular and chemical contrast. In skin, MPM contrast is derived from second harmonic generation (SHG) of collagen and two-photon excited fluorescence (TPEF) of autofluorescent co-factors NAD(P)H and FAD, elastin, keratin, and melanin. In addition to the specificity provided by the detection of the SHG signal from collagen, MPM can visualize specific skin fluorophores based on their fluorescence lifetime detection. Over the past several years, our group and others have demonstrated the MPM strong potential for a broad range of applications from advancing the understanding of skin biology to non-invasive diagnosis of skin diseases and monitoring therapy effects. However, a routine implementation of this technology in clinical research and practice requires advancing the instrumentation to allow for easier access by clinicians and for more efficient imaging in terms of speed and scanning area. Our group recently introduced benchtop innovations and developed a fast large area multiphoton exoscope (FLAME) that can provide rapid, real-time, depth-resolved images of skin, over macroscopic areas (cm-scale) with microscopic resolution (0.5-1m) and chemical contrast (selective detection of melanin). This presentation will highlight the latest advances in the FLAME development, including its conversion into a compact, portable device, highly optimized for clinical skin imaging with enhanced sensitivity and specificity. The technical abilities of this imaging platform will be demonstrated along with the results of a study that establishes clinical safety and imaging performance in 20 volunteers with normal skin. The results emphasize the significance of macroscopic imaging in the context of skin heterogeneity in terms of pigment distribution and dermal photodamage.
Our group has recently reported on the development of a fast large area multiphoton exoscope (FLAME) for rapid macroscopic imaging with microscopic resolution of human skin. This imaging platform is based on multiphoton microscopy (MPM) that provide label-free, depth-resolved images by using femtosecond laser pulses. Our goal is to use FLAME for in vivo skin imaging to address unmet needs in clinical research and practice of dermatology. This work evaluates the photodamage threshold induced by the excitation laser for the full Fitzpatrick scale of human skin. The optimum laser power and scanning regimes for safe imaging determined by this study will be used for further in vivo clinical studies.
Our group and others have demonstrated the strong potential of the multiphoton microscopy for a broad range of applications from advancing the understanding of skin biology to non-invasive diagnosis of skin diseases and monitoring therapy effects. We have recently reported on a fast large area multiphoton exoscope for rapidly mapping out macroscopic tissue areas with microscopic resolution and enhanced contrast for selective melanin detection. We will describe the technical abilities of this instrument and demonstrate its feasibility for early melanoma diagnosis based on a pilot study on ex-vivo and in-vivo imaging of pigmented lesions suspicious of melanoma in human skin.
In vivo multiphoton microscopy (MPM) provides non-invasive imaging of living tissues in their native state. Melanin, a fluorophore found in the skin, plays a significant role in various physiological and pathological processes. Quantification of cutaneous melanin provides basis for efficient treatment of pigmentary skin disorders and for differentiating melanoma from benign pigmented lesions. MPM imaging has a potential for non-invasive cutaneous melanin evaluation. We recently reported on the development of a fast large area multiphoton exoscope (FLAME) for in vivo macroscopic imaging with microscopic resolution and enhanced molecular contrast for selective melanin detection. In this work, we demonstrate the benefit of sampling a large volume, using FLAME in vivo, for enhancing the accuracy of the melanin content assessment in human skin.
Multiphoton microscopy enables sub-micron resolution, label-free structural and functional imaging of living tissues with contrast from multiple modalities, including second harmonic generation and two-photon excited fluorescence. We developed a fast, large area multiphoton exoscope (FLAME) portable system with enhanced label-free molecular contrast for macroscopic imaging of human skin with microscopic resolution. It combines optical and mechanical scanning mechanisms with deep learning image restoration to produce 3D sub-cellular resolution images that encompass sub-mm2 to cm2 scale areas of tissue within minutes. We demonstrate the performance and utility of the instrument by fast ex vivo and in vivo imaging of human skin.
KEYWORDS: Skin, In vivo imaging, Associative arrays, Tumors, Melanoma, Image resolution, Diagnostics, Denoising, Convolutional neural networks, Chemical elements
Multiphoton microscopy can provide sub-micron resolution images of living tissues in their
native environment with chemical contrast. We recently reported on a fast large area multiphoton
exoscope (FLAME) for rapidly mapping out macroscopic tissue areas (cm-scale) with microscopic
resolution. In this presentation we demonstrate the imaging capability and the clinical utility of this
system by performing a pilot study on ex vivo imaging of benign and malignant pigmented lesions of
human skin. We identify morphological features such as cytological atypia, lentiginous hyperplasia,
migration of melanocytes and demonstrate the value of sampling large tissue volumes for capturing the
lesion heterogeneity.
Vitiligo is a disorder characterized by the loss of skin pigment due to the autoimmune destruction of epidermal melanocytes. Treatments that aim to repigment vitiligo regions are long, incomplete, and suffer from a highly variable success rate. This study aims to use non-invasive, depth-resolved, label-free, two-photon detection of reduced nicotinamide adenine dinucleotide (NADH) to quantitatively evaluate mitochondrial dynamics in vitiligo micrografting patients during the repigmentation process. This study indicates differences in the depth-dependent mitochondrial profiles between healthy and vitiligo tissues. This study also indicates that vitiligo regions responsive to treatment exhibit depth-dependent shifts in mitochondrial dynamics prior to repigmentation.
Vitiligo is an immune skin disorder consisting of depigmented skin patches caused by the destruction of epidermal melanocytes. Vitiligo treatment represents a clinical challenge since the re-pigmentation mechanism is not fully understood. In this pilot study, we employ in-vivo multiphoton microscopy to evaluate epidermal keratinocyte metabolic state before and during treatment, in-vivo reflectance confocal microscopy to track melanocyte migration after treatment initiation, and single cell transcriptomics to identify unique cell populations more abundant in stable vitiligo lesions compared to normal skin. The findings provide insights into the role of certain cell populations in the viability of micro-grafting treatments.
Vitiligo is characterized by white patches on skin due to the loss of melanocytes. Treatments are not uniformly successful and re-pigmentation is rarely complete. Optical laser scanning microscopy techniques have great potential to advance our understanding of the repigmentation process of vitiligo.
In this pilot study, we employ in-vivo multiphoton microscopy (MPM) to assess potential changes in the metabolic state of epidermal keratinocytes involved in vitiligo before and throughout treatment, and in-vivo reflectance confocal microscopy (RCM) to assess the initiation of the re-pigmentation process and monitor wound healing after micro-grafting treatment.
Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Recent advances of MPM in clinical skin imaging demonstrated the unique potential of this technology as a label-free research and clinical tool for a broad area of applications such as melanoma and non-melanoma skin cancer detection, monitoring pigmentary skin disorders, characterizing keratinocyte metabolism, etc. In this contribution we demonstrate the ability of this microscope to provide sub-micrometer resolution ex-vivo images of large areas of skin tissue (up to 5x5 mm2) in <1 minute. We demonstrate the importance of high-speed, high-resolution mesoscopic imaging on cancerous skin tissues that present heterogeneous morphology to show the ability of the instrument to capture both benign and malignant areas of the lesion.
Vitiligo is a skin condition in which pigment-producing cells are removed by the immune system, leading to patches of white skin on different parts of the body. Treatments, including UVB light therapy and skin micro-grafting, may lead to repigmentation of the skin; however, treatments are not uniformly successful, and it is currently unclear why some vitiligo areas repigment more rapidly than others. An optical imaging technique that allows non-invasive visualization of melanocytic activity in skin may advance the knowledge about this skin condition and help understand treatment impact. In this pilot study, we employ in-vivo multiphoton microscopy (MPM) to evaluate architectural and structural features of the melanocytes that repigment vitiligo skin. MPM is a nonlinear laser scanning microscopy technique that features sub-cellular resolution and label-free molecular contrast. MPM contrast in skin is derived from two-photon excited fluorescence of NADH/FAD+, keratin, melanin, and elastin, and second-harmonic generation of collagen. We employed a clinical MPM tomograph (MPTflex, JenLab, Germany) to image vitiligo and adjacent normal areas in 10 patients undergoing treatment. The treatment consisted of either UVB light therapy or skin micro-grafting treatment followed by UVB light therapy. We visualized pigment producing melanocytes near hair follicles, migrating melanocytes within the human epidermis, newly pigmented keratinocytes in the basal layer, and epidermal melanin granules. The overarching goal is to use this technology to better define the phenotypic characteristics of migrating melanocytes in the hope of improving transplantation therapies for vitiligo.
Treatment and management of alopecia are highly determined by an accurate diagnosis, which can be challenging due to the lack of methods to properly visualize hair follicles. Current standard diagnosis is based on dermoscopy for non-scarring alopecia and scalp biopsy for scarring types of alopecia. Dermoscopy can be inconclusive, while biopsy is a painful procedure. In this study, we used a clinical tomograph based on multiphoton microscopy (MPM) to non-invasively image the scalp of 5 healthy subjects and of 12 patients affected by non-scarring alopecia (androgenetic and areata) and scarring alopecia (frontal fibrosing). MPM is capable of non-invasive in vivo imaging of follicular structures in human scalp including hair shafts, hair follicles and sebaceous glands via two-photon excited fluorescence (TPEF) from keratin and NADH/FAD and of the papillary dermis surrounding the hair follicles through second harmonic generation (SHG) from collagen and TPEF from elastin fibers. In normal and non-scarring alopecia patients, MPM often identified presence of sebaceous glands associated with hair follicles, while MPM images of scarring alopecia were characterized by miniaturization of hair follicles as well as by presence of macrophages and lymphocytes surrounding hair follicles. A quantitative analysis involving measurement of hair follicle diameter sizes showed they were significantly smaller in scarring comparing to non-scarring alopecia patients and to normal scalp subjects (p < 0.043). This study shows, in a limited number of patients, that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia.
Type 1diabetes (T1D) is an autoimmune disorder that occurs due to the rapid destruction of insulin-producing beta cells, leading to insulin deficiency and the inability to regulate blood glucose levels and leads to destructive secondary complications. Advanced glycation end (AGEs) products, the result of the cross-linking of reducing sugars and proteins within the tissues, are one of the key causes of major complications associated with diabetes such as renal failure, blindness, nerve damage and vascular changes. Non-invasive techniques to detect AGEs are important for preventing the harmful effects of AGEs during diabetes mellitus.
In this study, we utilized multiphoton microscopy to image biopsies taken from control rats and compared them to biopsies taken from streptozotocin (STZ) induced adult male diabetic rats. This was done at two and four weeks after the induction of hyperglycemia (>400 mg/dL) specifically to evaluate the effects of glycation on collagen. We chose to use an in-situ multiphoton microscopy method that combines multiphoton auto-florescence (AF) and second harmonic generation (SHG) to detect the microscopic influence of glycation.
Initial results show high auto-florescence levels were present on the collagen, as a result of the accumulation of AGEs only two weeks after the STZ injection and considerably higher levels were present four weeks after the STZ injection. Future projects could involve evaluating advanced glycation end products in a clinical trial of diabetic patients.
We use a multiphoton microscopy (MPM)-based clinical microscope (MPTflex, JenLab, Germany) to describe changes in human skin following treatment with a fractional non-ablative laser (PicoWay, Candela).
The treatment was based on a fractionated picosecond Nd:YAG laser (1064 and 532nm, 3mJ and 1.5mJ (no attenuation), respectively maximum energy/pulse, 100 microbeams/6mmx6mm). Improvements in skin appearance resulting from treatment with this laser have been noted but optimizing the efficacy depends on a thorough understanding of the specific skin response to treatment.
MPM is a nonlinear laser scanning microscopy technique that features sub-cellular resolution and label-free molecular contrast. MPM contrast in skin is derived from second-harmonic generation of collagen and two-photon excited fluorescence of NADH/FAD+, elastin, keratin, melanin.
In this pilot study, two areas on the arm of a volunteer (skin type II) were treated with the picoWay laser (1064nm, 3mJ; 532nm, 1.5mJ; 1pass). The skin response to treatment was imaged in-vivo at 8 time points over the following 4 weeks. MPM revealed micro-injuries present in epidermis. Damaged individual cells were distinguished after 3h and 24h from treatment with both wavelengths. Pigmented cells were particularly damaged in the process, suggesting that melanin is the main absorber and the primary target for laser induced optical breakdown. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. These results represent the groundwork for future longitudinal studies on expanded number of subjects to understand the response to treatment in different skin types at different laser parameters, critical factors in optimizing treatment outcomes.
We report on a direct comparison between Ti:Sapphire and Yb fiber lasers for depth-resolved label-free multimodal imaging of human skin. We found that the penetration depth achieved with the Yb laser was 80% greater than for the Ti:Sapphire. Third harmonic generation (THG) imaging with Yb laser excitation provides additional information about skin structure. Our results indicate the potential of fiber-based laser systems for moving into clinical use.
Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance in general, especially when they start to progress towards malignancy. Large field of view (FOV) and automatic translation of the imaging area are critical in the assessment of the entire lesion. Imaging of limited FOVs of the lesion can easily result in false negative diagnosis. We present a multiphoton microscope based on two-photon excited fluorescence and second-harmonic generation that images FOVs of about 0.8 mm2 (without stitching adjacent FOVs) at speeds of 10 frames/second (800 x 800 pixels) with lateral and axial resolutions of 0.5 μm and 2.5 μm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, relay optics, a beam expander and a high NA objective lens. We optimized the system based on the Olympus 25x, 1.05NA water immersion lens, that features a long working distance of 1 mm. Proper tailoring of the beam expander, which consists of the scan and tube lens elements, enables scaling of the FOV. The design criteria include a flat wavefront of the beam, minimum field curvature, and suppressed spherical aberrations. All aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide FOVs in normal human skin.
Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second‐harmonic generation (SHG) and third‐harmonic generation images from ex vivo human skin and showed that a sub‐40 fs, 1060‐nm Yb‐fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber‐based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low‐barrier‐to‐access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.
The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between ∼5% (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ∼30–65 μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R2=0.8895). SFDS melanin distribution thickness is correlated to MPM values (R2=0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.
In vivo imaging of pigmented lesions in human skin was performed with a clinical multiphoton microscopy (MPM)-based tomograph (MPTflex, JenLab, Germany). Two-photon excited fluorescence was used for visualizing endogenous fluorophores such as NADH/FAD, keratin, melanin in the epidermal cells and elastin fibers in the dermis. Collagen fibers were imaged by second harmonic generation. Our study involved in vivo imaging of benign melanocytic nevi,
atypical nevi and melanoma. The goal of this preliminary study was to identify in vivo the characteristic features and
their frequency in pigmented lesions at different stages (benign, atypical and malignant) and to evaluate the ability of in
vivo MPM to distinguish atypical nevi from melanoma. Comparison with histopathology was performed for the biopsied
lesions. Benign melanocytic nevi were characterized by the presence of nevus cell nests at the epidermal-dermal
junction. In atypical nevi, features such as lentiginous hyperplasia, acanthosis and architectural disorder were imaged.
Cytological atypia was present in all the melanoma lesions imaged, showing the strongest correlation with malignancy. The MPM images demonstrated very good correlation with corresponding histological images, suggesting that MPM
could be a promising tool for in vivo non-invasive pigmented lesion diagnosis, particularly distinguishing atypical nevi
from melanoma.
Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.
The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent
advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant
melanoma diagnostics.
The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and
pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation.
We acquired TPEF emission spectra (λex=1000 nm) of melanin from several specimens, including human hair, malignant
melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in
five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted
from the eumelanin with broad maximum at 640-680nm.
We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm.
The measured OMI for a melanoma cell line MNT-1 was 1.6±0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown)
showed an anticipated change in melanins production ratio and had OMI of 0.55±0.05 and 0.17±0.02,
respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells
layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05
and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI.
These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization
both in vitro and in vivo, including pigmented lesions.
KEYWORDS: Tissues, CARS tomography, Signal detection, Single mode fibers, Femtosecond phenomena, Signal generators, Picosecond phenomena, Silica, In vivo imaging, Skin
We demonstrate a fiber-based probe for maximum collection of the Coherent
anti-Stokes Raman Scattering (CARS) signal in biological tissues. We discuss the design
challenges including capturing the back-scattered forward generated CARS signal in the
sample and the effects of fiber nonlinearities on the propagating pulses. Three different
biological tissues were imaged in vitro in order to assess the performance of our fiberdelivered
probe for CARS imaging, a tool which we consider an important advance
towards label-free, in vivo probing of superficial tissues.
We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 µm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-µm excitation in nonlinear optical microscopy.
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