3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

Neurite growth acceleration of adult Dorsal Root Ganglion neurons illuminated by low‐level Light Emitting Diode light at 645 nm

The effect of a 645 nm Light Emitting Diode (LED) light irradiation on the neurite growth velocity of adult Dorsal Root Ganglion (DRG) neurons with peripheral axon injury 4–10 days before plating and without previous injury was investigated. The real amount of light reaching the neurons was calculated by taking into account the optical characteristics of the light source and of media in the light path. The knowledge of these parameters is essential to be able to compare results of the literature and a way to reduce inconsistencies. We found that 4 min irradiation of a mean irradiance of 11.3 mW/cm² (corresponding to an actual irradiance reaching the neurons of 83 mW/cm²) induced a 1.6‐fold neurite growth acceleration on non‐injured neurons and on axotomized neurons. Although the axotomized neurons were naturally already in a rapid regeneration process, an enhancement was found to occur while irradiating with the LED light, which may be promising for therapy applications.

Dorsal Root Ganglion neurons ( A ) without previous injury and ( B ) subjected to a conditioning injury.     

Light‐emitting diode irradiation using 660 nm promotes human fibroblast HSP90 expression and changes cellular activity and morphology

We evaluated changes in cell viability and morphology in response to low‐level light irradiation and underlying variations in the levels of heat shock proteins (HSPs). Human fibroblasts were irradiated with a light‐emitting diode (LED) array at 660 nm (50 mW for 15, 30, and 60 minutes). Cell viability and morphological changes were evaluated via epifluorescence analysis; we also assessed cell viability and length changes. The expression levels of adenosine triphosphate (ATP) and various HSPs (HSP27, 60, 70, and 90) were analyzed by immunohistochemical staining, Western blotting and microarray analysis. After LED irradiation, cellular viability and morphology changed. Of the several HSPs analyzed, the HSP90 level increased significantly, suggesting that this protein played roles in the morphological and cellular changes. Thus, low‐level irradiation triggered cellular changes mediated by increased HSP90 expression; this may explain why skin irradiation enhances wound‐healing.     

Sex,but not skin tone affects penetration of red‐light (660 nm) through sites susceptible to sports injury in lean live and cadaveric tissues

Red‐light treatment is emerging as a novel therapy for promoting tissue recovery but data on red‐light penetration through human tissues are lacking. We aimed to: (1) determine the effect of light irradiance, tissue thickness, skin tone, sex and bone/muscle content on 660 nm light penetration through common sites of sports injuries, and (2) establish if cadaver tissues serve as a useful model for predicting red‐light penetration in live tissues. Live and cadaver human tissues were exposed to 660 nm light at locations across the skull, spinal cord and upper and lower limbs. Red‐light was produced by a light emitting diode array of various irradiances (15‐500 mW/cm²) and measured by a light‐probe positioned on the tissue surface opposite to the light emitting diodes. 100 mW/cm² successfully penetrated tissue <50 mm thick; a disproportionate irradiance increase was required to achieve deeper penetration. Penetration was unaffected by skin tone, increased with irradiance and relative bone/muscle composition, and decreased with greater tissue thickness and in males. Live and cadaveric tissue penetration did not differ statistically for tissues <50 mm but cadavers required more red‐light to penetrate >50 mm. These results assist clinicians and researchers in determining red‐light treatment intensities for penetrating human tissues.      

Light‐emitting diode therapy in exercise‐trained mice increases muscle performance,cytochrome c oxidase activity,ATP and cell proliferation

Light‐emitting diode therapy (LEDT) applied over the leg, gluteus and lower‐back muscles of mice using a LED cluster (630 nm and 850 nm, 80 mW/cm², 7.2 J/cm²) increased muscle performance (repetitive climbing of a ladder carrying a water‐filled tube attached to the tail), ATP and mitochondrial metabolism; oxidative stress and proliferative myocyte markers in mice subjected to acute and progressive strength training. Six bi‐daily training sessions LEDT‐After and LEDT‐Before‐After regimens more than doubled muscle performance and increased ATP more than tenfold. The effectiveness of LEDT on improving muscle performance and recovery suggest applicability for high performance sports and in training programs.

Positioning of the mice and light‐emitting diode therapy (LEDT) applied on mouse legs, gluteus and lower‐back muscles without contact.     

Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca²⁺ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.     

Polarization‐multiplexed,dual‐beam swept source optical coherence tomography angiography

A polarization‐multiplexed, dual‐beam setup is proposed to expand the field of view (FOV) for a swept source optical coherence tomography angiography (OCTA) system. This method used a Wollaston prism to split sample path light into 2 orthogonal‐polarized beams. This allowed 2 beams to shine on the cornea at an angle separation of ~14°, which led to a separation of ~4.2 mm on the retina. A 3‐mm glass plate was inserted into one of the beam paths to set a constant path length difference between the 2 polarized beams so the interferogram from the 2 beams are coded at different frequency bands. The resulting OCTA images from the 2 beams were coded with a depth separation of ~2 mm. A total of 5 × 5 mm² angiograms from the 2 beams were obtained simultaneously in 4 seconds. The 2 angiograms then were montaged to get a wider FOV of ~5 × 9.2 mm².      

Photodynamic therapy associating Photogem® and blue LED on L929 and MDPC‐23 cell culture

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem® associated to blue LED (light‐emitting diode) on L929 and MDPC‐23 cell cultures, 30000 cells/cm² were seeded in 24‐well plates for 48 h, incubated with Photogem® (10, 25 or 50 mg/l) and irradiated with an LED source (460±3 nm; 22 mW/cm²) at two energy densities (25.5 or 37.5 J/cm²). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet's post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT‐induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem® concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT‐treated cells presented an altered morphology with ill‐defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem® or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.     

Potentials for prolonging shelf‐life of platelets by near infrared low‐level light

Platelets are uniquely stored at room temperature, during which they gradually loss their quality owing to deteriorating functions of mitochondria over time. Given the well‐documented beneficial effect of near infrared low‐level light (LLL) on mitochondrial functions, we explored a potential for LLL to protect mitochondrial function and extend the shelf‐life of platelets beyond the current 5 days. We found that exposure of a platelet‐containing storage bag to 830 nm light‐emitting diode (LED) light at 0.5 J/cm² prior to storage could significantly retain a pH value and viability of the platelets stored for 8 days with improved quality compared to those stored similarly for 5 days in controls. The LLL inhibited reactive oxygen species (ROS) and lactate production, while sustaining ATP synthesis and mitochondrial membrane potential and morphology in the stored platelets. It also sustained aggregation capacity and in vivo survival of stored platelets, concomitant with no significant activation, as suggested by similar CD62p expression and enhanced agonist‐induced aggregation and recovery following infusion in the presence compared to absence of LLL treatment. This simple, additive‐free, cost‐effective, noninvasive approach can be readily incorporated into the current platelet storage system to potentially improve quality of stored platelets.      

Bifunctional Moth‐Eye Nanopatterned Dye‐Sensitized Solar Cells: Light‐Harvesting and Self‐Cleaning Effects

A nanopatterning technique using nanostamps that provides a facile process to create a nature‐inspired moth‐eye structure achieving high transmittance in the visible range as well as a self‐cleaning effect is reported. Commercially available perfluoropolyether (PFPE) and NOA63 as the mold resin and second replica mold material, respectively, play an important role in fabricating the structure. The structure is found to increase transmittance up to 82% at 540 nm and contact angle up to 150°, representing superhydrophobicity even without the aid of a fluorinated self‐assembled monolayer (SAM) coating. The resulting solid‐state dye‐sensitized solar cells (ssDSSCs) with moth‐eye structures show enhancement of efficiency to 7.3% at 100 mW cm^?2, which is among the highest values reported to date for N719 dye‐based ssDSSCs. This nature‐inspired nanopatterning process could be used for improving light harvesting in any type of photovoltaic cell, and it produces superhydrophobic surfaces, which in turn lead to self‐cleaning for long‐term stability.     

Enhanced germicidal effects of pulsed UV‐LED irradiation on biofilms

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low‐frequency pulsed ultraviolet A (UVA)‐light‐emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA‐LED irradiation (365 nm, 0·28 mW cm^?2, in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony‐forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5‐min irradiation, over 90% of viable micro‐organisms in biofilms had been killed, and pulsed irradiation (1–1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro‐organisms in biofilm (>99·9%), and 20‐min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH^?), significantly protected viable micro‐organisms in biofilms from UVA‐LED irradiation. Conclusions: The study demonstrated that pulsed UVA‐LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5‐min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low‐frequency pulsed UVA‐LED system to be applied to pathogenic biofilms for disinfection.     

Visible light regulates neurite outgrowth of nerve cells

The neurite outgrowth of PC12 cells on collagen-coated glass plates under light emitting diode (LED) irradiation at several wavelengths (i.e., 455, 470, 525, 600, 630, 880 and 945 nm) was investigated. No neurite outgrowth was observed during cultivation under irradiation from the lamp of an inverted light microscope through filters (yielding mixed light at ca. 525 nm and more than 800 nm), whereas neurite outgrowth was observed during cultivation in the dark. When these cells were irradiated with monochromatic LED light, neurite outgrowth was slightly, but not completely, suppressed at 455, 525, 600, 630, 880 and 945 nm, as was observed in the case of mixed light. Long connected neuronal outgrowths (e.g., 3 mm length) were observed with LED light at 470 nm and 1.8 mW/cm² intensity. No such outgrowths were observed at other LED light wavelengths (i.e., 455, 525, 600, 630, 880 and 945 nm). Irradiation at 470 nm may have caused specific responses to transductional signals in these cells that led to the connection of neuronal outgrowths between cells. Not only suppressed neurite outgrowth but also long connected neurite outgrowths were observed when PC12 cells were cultured under several different wavelengths of light.     

Tunable fibre‐coupled multiphoton microscopy with a negative curvature fibre

Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.3 dB m^–1 over the range 600–850 nm and the GVD was –180 ± 70 fs² m^–1 at 800 nm. Using an average fibre output power of ～20 mW and pulse repetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmitted through a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersion compensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within 10% of low power values up to the maximum fibre output power achievable with the laser system used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupled to a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengths from 740 nm to 860 nm without any need for adjustments to the set‐up.

     

Nanodiamond‐Embedded p‐Type Copper(I) Oxide Nanocrystals for Broad‐Spectrum Photocatalytic Hydrogen Evolution

Copper(I) oxide (Cu₂O) is an attractive photocatalyst because of its abundance, low toxicity, environmental compatibility, and narrow direct band gap, which allows efficient light harvesting. However, Cu₂O exhibits poor photocatalytic performance and photostability because of its short electron diffusion length and low hole mobility. Here, it is demonstrated that nanodiamond (ND) can greatly improve the photocatalytic hydrogen evolution reaction (HER) of the p‐type photocatalyst Cu₂O nanocrystals by nanocomposition. Compared with pure Cu₂O nanocrystals, this composite shows a tremendous improvement in HER performance and photostability. HER rates of 100.0 mg NDs‐Cu₂O nanocrystals are 1597 and 824 under the simulated solar light irradiation (AM 1.5, 100 mW cm^?2) and visible light irradiation (420–760 nm, 77.5 mW cm^?2), respectively. The solar‐to‐hydrogen conversion efficiency of this composite is 0.85%, which is nearly ten times higher than that of pure Cu₂O. The quantum efficiency of the composite is high, with values of 0.17% at and 0.23% at . The broad spectral response of ND provides numerous carriers for the subsequent reactions. The electron‐donating ability of ND and suitable band structures of the two components promote electron injection from ND to Cu₂O. These results suggest the broad applicability of ND to ameliorate the photoelectric properties of semiconductors.     

Two‐photon imaging of fiber‐coupled neurons

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two‐photon imaging is shown to enable a quantitative in situ analysis of such fiber–single‐neuron coupling in the live brain of transgenic mice. Fiber‐optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two‐photon imaging. These results pave the way for fiber‐optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber‐optic single‐neuron stimulation into the optical imaging framework.

     

Blue excitable green emitting Ce3+ doped CaS phosphor for w‐LEDs

CaS:Ce³⁺ is an efficient green‐emitting (535 nm) phosphor, excitable with blue light (450–470 nm) and was synthesized via a solid‐state reaction method by heating under a reducing atmosphere. The luminescent properties, photoluminescent (PL) excitation and emission of the phosphor were analyzed by spectrofluorophotometry. The excitation and emission peaks of the CaS:Ce³⁺ phosphor lay in the visible region, which made them relevant for light‐emitting diode (LED) application for the generation of white light. Judd‐Oflet parameters were calculated and revealed that green light emitted upon blue illumination. The prepared phosphor had strong blue absorption at 470 nm and a broad green emission band range from 490–590 nm with the peak at 537 nm. The characteristics of the CaS:Ce³⁺ phosphor make it suitable for use as a wavelength tunable green emitting phosphor for three band white LEDs pumped by a blue LED (470 nm). The Commission International de l'Eclairage co‐ordinates were calculated by a spectrophotometric method using the spectral energy distribution (0.304, 0.526) and confirm the green emission. The potential application of this phosphor is as a phosphor‐converted white light‐emitting diode. Copyright © 2015 John Wiley & Sons, Ltd.