Spectral morphological analysis of skin lesions with a polarization multispectral dermoscope.

Dermoscopy is the conventional technique used for the clinical inspection of human skin lesions. However, the identification of diagnostically relevant morphologies can become a complex task. We report on the development of a polarization multispectral dermoscope for the in vivo imaging of skin lesions. Linearly polarized illumination at three distinct spectral regions (470, 530 and 625 nm), is performed by high luminance LEDs. Processing of the acquired images, by means of spectral and polarization filtering, produces new contrast images, each one specific for melanin absorption, hemoglobin absorption, and single scattering. Analysis of such images could facilitate the identification of pathological morphologies.

Blue-LED-Light Photobiomodulation of Inflammatory Responses and New Tissue Formation in Mouse-Skin Wounds.

Background: Recent studies evidence that blue-LED-light irradiation can modulate cell responses in the wound healing process within 24 h from treatment. This study aims to investigate blue-light (410-430 nm) photobiomodulation used in a murine wound model within six days post-treatment. Methods: A superficial wound was made in 30 CD1 male mice. The injuries were treated with a blue LED light (20.6 J/cm2), and biopsies were collected at 24, 72, and 144 h. Histology, fluorescence analysis, and advanced microscopy techniques were used. Results: We can observe an increase in the cellular infiltrate response, and in mast-cell density and their degranulation index correlated to the expression of the major histocompatibility complex after 24 h. Furthermore, after six days, the vessel density increases with the expression of the platelet-derived growth factor in the mast cells. Finally, collagen deposition and morphology in the treated wounds appear more similar to unwounded skin. Conclusions: Blue-light photobiomodulation stimulates several cellular processes that are finely coordinated by mast cells, leading to more rapid wound healing and a better-recovered skin morphology.

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.

Experimental Study on Blue Light Interaction with Human Keloid-Derived Fibroblasts.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm2 power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm2) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm2) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.

Confocal multispot microscope for fast and deep imaging in semicleared tissues.

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-µm deep.

Three-dimensional color object visualization and recognition using multi-wavelength computational holography.

In this paper, we address 3D object visualization and recognition with multi-wavelength digital holography. Color features of 3D objects are obtained by the multiple-wavelengths. Perfect superimposition technique generates reconstructed images of the same size. Statistical pattern recognition techniques: principal component analysis and mixture discriminant analysis analyze multi-spectral information in the reconstructed images. Class-conditional probability density functions are estimated during the training process. Maximum likelihood decision rule categorizes unlabeled images into one of trained-classes. It is shown that a small number of training images is sufficient for the color object classification.

Observation of an improved healing process in superficial skin wounds after irradiation with a blue-LED haemostatic device.

The healing process of superficial skin wounds treated with a blue-LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue-LED device, while the other two are left to naturally recover. Visual observations, non-linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better-recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content. Blue LED induced photothermal effect on superficial abrasions.

Extended focused image in microscopy by digital Holography.

In microscopy, high magnifications are achievable for investigating micro-objects but the paradigm is that higher is the required magnification, lower is the depth of focus. For an object having a three-dimensional (3D) complex shape only a portion of it appears in good focus to the observer who is essentially looking at a single image plane. Actually, two approaches exist to obtain an extended focused image, both having severe limitations since the first requires mechanical scanning while the other one requires specially designed optics. We demonstrate that an extended focused image of an object can be obtained through digital holography without any mechanical scanning or special optical components. The conceptual novelty of the proposed approach lies in the fact that it is possible to completely exploit the unique feature of DH in extracting all the information content stored in hologram, amplitude and phase, to extend the depth of focus.

On the origin of internal field in Lithium Niobate crystals directly observed by digital holography.

We show the defect dependence of the internal field in Lithium Niobate using a full-field interferometric method and demonstrate that it can be directly measured on some clusters of defects embedded in a stoichiometric matrix. Results show that the value of the internal field grows in proximity of defects and vanishes far from them, which addresses the long-standing issue about its origin in Lithium Niobate crystal.

Multispot multiphoton Ca²âº imaging in acute myocardial slices.

Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca²âº dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca²âº dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca²âº imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca²âº dynamics in different cells in the tissue. We recorded local Ca²âº release events including macrosparks, travelling waves, and rotors.

In-situ visualization, monitoring and analysis of electric field domain reversal process in ferroelectric crystals by digital holography.

In-situ monitoring of domain reversal in congruent lithium niobate by a digital holographic technique is described. While the ferroelectric polarization is reversed by electric field poling, the two-dimensional distribution of the phase shift, due mainly to the linear electro-optic and piezoelectric effects, is measured and visualized. Digital holography is used to reconstruct both amplitude and phase of the wavefield transmitted by the sample to reveal the phase shift induced by adjacent reversed domains during the poling. The resulting movies of both amplitude and phase maps, for in-situ visualization of domain pattern formation, are shown. The possibility of using the technique as tool for monitoring in real-time the periodic poling of patterned samples is discussed.

In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy.

Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 µm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 µm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments.

Three-dimensional image fusion by use of multiwavelength digital holography.

We present three-dimensional (3D) image fusion by use of digital holography. We demonstrate experimentally that, through the image fusion technique with multiresolution wavelet decomposition, it is possible to increase the details and contrast of 3D reconstructed images obtained by multiwavelength digital holography. Although there is substantial activity in the fields of image fusion and holography, to the best of our knowledge, this is the first report of 3D image fusion by use of digital holography.

Controlling image size as a function of distance and wavelength in Fresnel-transform reconstruction of digital holograms.

A method for controlling the size of amplitude and phase images reconstructed from digital holograms by the Fresnel-transform method is proposed and demonstrated. The method can provide a constant reconstruction pixel width in the reconstructed image plane, independent of the recording and reconstruction distance. The proposed method makes it possible to maintain the size of an object for a sequence of digital holograms recorded at different distances and, therefore, to subtract phase maps for an object recorded at different distances. Furthermore, the method solves the problem of superimposition in multiwavelength digital holography for color display and holographic interferometry applications.