Optical Effects of Focused Fractional Nanosecond 1064-nm Nd:YAG Laser: Techniques of Application on Human Skin.

BACKGROUND AND OBJECTIVES: Considering the pulse widths of picosecond and nanosecond lasers used in cutaneous laser surgery differ by approximately one order of magnitude, can nanosecond lasers produce the optical effect in human skin similar to laser-induced optical breakdown (LIOB) caused by picosecond lasers? METHODS: Cutaneous changes induced by a focused fractional nanosecond 1064-nm Nd:YAG laser were evaluated by VISIA-CR imaging, histological examination, and harmonic generation microscopy (HGM). RESULTS: A focused fractional nanosecond 1064-nm Nd:YAG laser can generate epidermal vacuoles or dermal cavities similar to the phenomenon of LIOB produced by picosecond lasers. The location and extent of photodisruption can be controlled by the laser fluence and focus depth. Moreover, laser-induced shock wave propagation and thermal degeneration of papillary collagen can be observed by HGM imaging. CONCLUSION: Focused fractional nanosecond lasers can produce an optical effect on human skin similar to LIOB caused by picosecond lasers. With techniques of application, the treatment can induce epidermal and dermal repair mechanisms in a tunable fashion to improve skin texture, wrinkles, scars, and dyspigmentation, without disrupting the epidermal surface.

Characterization of picosecond laser-induced optical breakdown using harmonic generation microscopy.

BACKGROUND AND OBJECTIVES: By creating microinjuries usually confined to the epidermis, a fractional picosecond 1064-nm Nd:YAG laser that delivers an array of highly focused beamlets can be effectively used for facial rejuvenation or resurfacing. However, the mechanism of dermal remodeling underlying this nonablative treatment remains unclear. METHODS: Five participants having skin phototype III-IV were recruited for intervention using a fractional picosecond 1064-nm Nd:YAG laser system equipped with a holographic diffractive beam-splitting optic. The laser-induced histopathological changes on human skin were examined in vivo using a harmonic generation microscopy (HGM), visualizing second harmonic generation (SHG), and third harmonic generation (THG) contrasts dichromatically. SHG refers for collagen distribution, while THG represents for epidermal components in the HGM signal. RESULTS: Histological hematoxylin and eosin staining and in vivo HGM imaging studies revealed the presence of epidermal vacuoles below the stratum granulosum along with keratinocyte degeneration or cytolysis. In addition to the epidermal vacuoles, HGM imaging exclusively demonstrated laser-induced shock wave propagation arranged as a THG-bright concentric pattern in the epidermis and loss of SHG signals in the papillary dermis immediately beneath the epidermal vacuoles. CONCLUSIONS: Alongside generating epidermal vacuoles, the fractional picosecond 1064-nm Nd:YAG laser induced collagen changes. These collagen changes may lead to dermal remodeling and neocollagenesis underlying the fractional picosecond laser treatment.

A dose response relationship between accelerometer assessed daily steps and depressive symptoms in older adults: a two-year cohort study.

OBJECTIVES: this study investigated the prospective associations of accelerometer assessed daily steps with subsequent depressive symptoms in older adults. METHODS: a 2-year prospective study was performed in the community. A total of 285 older adults ≥65 years (mean age = 74.5) attended the baseline assessment in 2012. The second wave of assessment was carried out in 2014 including 274 (96.1%) participants. Daily step counts were measured with a triaxial accelerometer (ActiGraph GT3X+), and participants were divided into three categories (<3,500, 3,500-6,999 and ≥ 7,000 steps/day). The 15-item Geriatric Depression Scale was used to measure depressive symptoms. Negative binomial regression models with multivariable adjustment for covariates (baseline depressive symptoms, accelerometer wear time, age, gender, education, chronic disease, activities of daily living) were conducted to examine the association between daily steps and subsequent depressive symptoms. RESULTS: each 1,000-step increase in daily walking was linearly associated with a reduced rate of subsequent depressive symptoms (rate ratio [RR] = 0.95, 95% confidence interval [CI] = 0.92-0.98). Participants with daily step count in 3,500-6,999 (RR = 0.84, 95% CI = 0.70-0.99) and ≥7,000 steps (RR = 0.71, 95% CI = 0.55-0.92) per day had fewer depressive symptoms at follow-up. Sensitivity analyses assessing confounding and reverse causation provided further support for the stability of our findings. CONCLUSION: older adults engaging in more daily steps had fewer depressive symptoms after 2 years. Even as few as 3,500-6,999 steps a day was associated with a protecting effect. Accumulating ≥7,000 steps a day could provide the greatest protection against depressive symptoms.

Presence of intralesional melanocytes as a histopathological feature of actinic keratosis based on in vivo harmonic generation microscopy in Asians.

BACKGROUND: Most patients with actinic keratosis (AK) present with more than one lesion. Although histopathological examination is the gold standard for diagnosing this condition, performing an invasive skin biopsy for each AK is impractical. Thus, this study aimed to identify AK's morphological characteristics based on harmonic generation microscopy (HGM). Moreover, the correlation between features observed using HGM and histopathological grading of AK was examined. METHODS: Lesions of seven patients were examined using HGM (n = 1, ex vivo and n = 6, in vivo), and histopathological examinations of the biopsy specimens were also performed. The features of each AK, based on HGM, were assessed and compared with corresponding standard histopathological findings. RESULTS: Using the histopathological findings as a standard reference, HGM's accuracy in detecting features of AK lesions, such as hyperkeratosis, epidermal thinning, abnormal architecture, and atypical honeycomb pattern, was 100%. Approximately five (72%) patients had similar histopathological grades. Moreover, based on HGM, except for one patient with grade 1 AK, six (85.71%) patients had lesions with intraepidermal dendritic cell-like cells, representing melanocytes. CONCLUSION: Harmonic generation microscopy can be used in vivo to provide critical diagnostic information with a resolution comparable to histopathological examination. In addition, intralesional melanocytes in AK, which may be correlated with disease severity, can be specifically enhanced using HGM.

Ultra-short photoacoustic pulse generation through hot electron pressure in two-dimensional electron gas.

Launching ultrashort femtosecond photoacoustic pulses with multi-terahertz bandwidth will find broad applications from fundamental acoustics in 2D materials and THz-acoustic and phonon spectroscopy to nondestructive detection in opaque materials with a sub-nanometer resolution. Here we report the generation of ultra-short 344 fs photoacoustic pulses with a 2.1 THz bandwidth from interfacial two-dimensional electron gas using optical femtosecond excitation. A comparison with simulation supports the dominant contribution of hot electron pressure and the ultrafast electron relaxation to produce pulsewidth shorter than the acoustic transit time across the electron wavefunction. Our simulation further indicates the possibility to generate <200 fs photoacoustic pulse.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope.

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Detection of malformations in sea urchin plutei exposed to mercuric chloride using different fluorescent techniques.

Embryos of Mediterranean sea urchin Paracentrotus lividus and subtropical Echinometra mathaei were exposed to 5,10, 15 and 20µgL(-1), and to 1, 2, 3 and 4µgL(-1) mercuric chloride (HgCl2), respectively. The effective concentration (EC50) inducing malformation in 50% of 4-arm pluteus stage (P4) was 16.14µgL(-1) for P. lividus and 2.41µgL(-1) for E. mathaei. Two-photon (TP), second (SHG) and third harmonic generation (THG) microscopy techniques, TUNEL staining, propidium iodide (PI) and Hoechst 33342 probes were used to detect light signals or to stain apoptotic and necrotic cells in fixed and alive plutei. Signals were detected differently in the two species: TP fluorescence, commonly associated with apoptotic cells, did not increase with increasing HgCl2 concentrations in P. lividus and in fact, the TUNEL did not reveal induction of apoptosis. PI fluorescence increased in P. lividus in a dose-dependent manner, suggesting a loss of cell permeability. In E. mathaei plutei TP fluorescence increased at increasing HgCl2 concentrations. THG microscopy revealed skeletal rods in both species. Different fluorescent techniques, used in this study, are proposed as early-warning systems to visualize malformations and physiological responses in sea urchin plutei.

Imaging Endogenous Bilirubins with Two-Photon Fluorescence of Bilirubin Dimers.

On the basis of an infrared femtosecond Cr:forsterite laser, we developed a semiquantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly differentiated hepatocellular carcinoma (HCC) tissues on average showed 3.7 times lower concentration of bilirubins than the corresponding nontumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than that of nontumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of nontumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.

Near-field sub-THz transmission-type image system for vessel imaging in-vivo.

A THz near-field transmission imaging system was successfully demonstrated to image the vessels inside the ears of nude mice in vivo. Wave-guided illumination and near-field scanning detection with a sub-wavelength aperture were applied. An operating frequency of 340 GHz was chosen to achieve a higher penetration depth in tissues with a reasonable signal-to-noise ratio. The near-field pattern of the power transmittance through the vessel was also numerically simulated and showed good correspondence to the measured results. The capability of the system for long-term monitoring in vivo was also demonstrated.

Pilot clinical study to investigate the human whole blood spectrum characteristics in the sub-THz region.

We have conducted a pilot clinical study to not only investigate the sub-THz spectra of ex-vivo fresh human whole blood of 28 patients following 8-hours fasting guideline, but also to find out the critical blood ingredients of which the concentration dominantly affects those sub-THz spectra. A great difference between the sub-THz absorption properties of human blood among different people was observed, while the difference can be up to ~15% of the averaged absorption coefficient of the 28 samples. Our pilot clinical study indicates that triglycerides and the number of red blood cells were two dominant factors to have significant negative correlation to the sub-THz absorption coefficients.

Third-harmonic generation microscopy reveals dental anatomy in ancient fossils.

Fossil teeth are primary tools in the study of vertebrate evolution, but standard imaging modalities have not been capable of providing high-quality images in dentin, the main component of teeth, owing to small refractive index differences in the fossilized dentin. Our first attempt to use third-harmonic generation (THG) microscopy in fossil teeth has yielded significant submicrometer level anatomy, with an unexpectedly strong signal contrasting fossilized tubules from the surrounding dentin. Comparison between fossilized and extant teeth of crocodilians reveals a consistent evolutionary signature through time, indicating the great significance of THG microscopy in the evolutionary studies of dental anatomy in fossil teeth.

Pore-size dependent THz absorption of nano-confined water.

We performed a THz absorption spectroscopy study on liquid water confined in mesoporous silica materials, MCM-41-S-18 and MCM-41-S-21, of two different pore sizes at room temperatures. We found that stronger confinement with a smaller pore size causes reduced THz absorption, indicating reduced water mobility due to confinement. Combined with recent theoretical studies showing that the microscopic structure of water inside the nanopores can be separated into a core water region and an interfacial water region, our spectroscopy analysis further reveals a bulk-water-like THz absorption behavior in the core water region and a solid-like THz absorption behavior in the interfacial water region.

Fractional thermolysis by bipolar radiofrequency facilitates cutaneous delivery of peptide and siRNA with minor loss of barrier function.

PURPOSE: In this study, we aimed to illustrate the utility of fractional radiofrequency (RF) that generated microchannels in the skin, allowing delivery of peptide and siRNA via the skin. The mechanisms involved in the correlation between macromolecule permeation and skin structure were also elucidated. METHODS: The morphology of the skin was examined by transmission electron microscopy (TEM), higher harmonic generation microscopy (HGM), and physiological factors. In vivo skin distribution of macromolecules was assessed by fluorescence and confocal microscopies. RESULTS: RF thermolysis selectively created an array of micropores deep into the epidermis without significant removal of the stratum corneum (SC). With energy of 30 mJ, a pore depth of 35 µm was achieved. The bipolar RF resulted in a 3-fold increase of transepidermal water loss (TEWL) compared with intact skin. The respective skin accumulation and flux of the peptide with a molecular weight (MW) of 2335 Da was 3- and 23-fold greater for the RF-treated group than for the non-treatment group. RF enhanced skin accumulation of siRNAs with MW of 10 and 15 kDa by 6.2- and 2.6-fold, respectively. Cutaneous penetration of the macromolecules with an MW of at least 40 kDa could be accomplished by RF. Confocal microscopy imaging revealed that RF could effectively deliver the peptide up to at least a 74-µm depth. The penetration depth of siRNA by RF irradiation was about 50 µm. CONCLUSIONS: The novel RF device efficiently delivered macromolecules into the skin while reserving SC layers to support some barrier functions. In this work, for the first time the assistance of fractional RF on peptide and siRNA transport was demonstrated.

Graphene-to-substrate energy transfer through out-of-plane longitudinal acoustic phonons.

Practically, graphene is often deposited on substrates. Given the major substrate-induced modification of properties and considerable energy transfer at the interface, the graphene-substrate interaction has been widely discussed. However, the proposed mechanisms were restricted to the two-dimensional (2D) plane and interface, while the energy conduction in the third dimension is hardly considered. Herein, we disclose the transfer of energy perpendicular to the interface of the combined system of the 2D graphene and the 3D base. More precisely, our observation of the energy dissipation of optically excited graphene via emitting out-of-plane longitudinal acoustic phonon into the substrate is presented. By applying nanoultrasonic spectroscopy with a piezoelectric nanolayer embedded in the substrate, we found that under photoexcitation by a femtosecond laser pulse graphene can emit longitudinal coherent acoustic phonons (CAPs) with frequencies over 1 THz into the substrate. In addition, the waveform of the CAP pulse infers that the photocarriers and sudden lattice heating in graphene caused modification of graphene-substrate bond and consequently generated longitudinal acoustic phonons in the substrate. The direct observation of this unexplored graphene-to-substrate vertical energy transfer channel can bring new insights into the understanding of the energy dissipation and limited transport properties of supported graphene.

Nonlinear photoacoustic microscopy via a loss modulation technique: from detection to imaging.

In order to achieve high-resolution deep-tissue imaging, multi-photon fluorescence microscopy and photoacoustic tomography had been proposed in the past two decades. However, combining the advantages of these two imaging systems to achieve optical-spatial resolution with an ultrasonic-penetration depth is still a field with challenges. In this paper, we investigate the detection of the two-photon photoacoustic ultrasound, and first demonstrate background-free two-photon photoacoustic imaging in a phantom sample. To generate the background-free two-photon photoacoustic signals, we used a high-repetition rate femtosecond laser to induce narrowband excitation. Combining a loss modulation technique, we successfully created a beating on the light intensity, which not only provides pure sinusoidal modulation, but also ensures the spectrum sensitivity and frequency selectivity. By using the lock-in detection, the power dependency experiment validates our methodology to frequency-select the source of the nonlinearity. This ensures our capability of measuring the background-free two-photon photoacoustic waves by detecting the 2nd order beating signal directly. Furthermore, by mixing the nanoparticles and fluorescence dyes as contrast agents, the two-photon photoacoustic signal was found to be enhanced and detected. In the end, we demonstrate subsurface two-photon photoacoustic bio-imaging based on the optical scanning mechanism inside phantom samples.

Gigahertz coherent guided acoustic phonons in AlN/GaN nanowire superlattices.

The generation of guided acoustic phonons in the GHz range in GaN/AlN superlattices grown atop a GaN nanowire is presented. Combined with a femtosecond laser, ultrafast pump-probe spectroscopy allows the generation and detection of guided acoustic phonons at different frequencies in the nanowire superlattices. The capability of the nanowire superlattices to be excellent detectors of acoustic phonons at specific frequencies is then used to observe the strong dispersion, as a result of nanoconfinement, of guided acoustic phonons after their propagation in the nanowire. The generation of high frequency coherent guided acoustic phonons could be useful not only to realize an acoustic transducer with a nanolateral size but also as a source to understand the thermal behavior of nanowires.

Deep learning-based photodamage reduction on harmonic generation microscope at low-level optical power.

The trade-off between high-quality images and cellular health in optical bioimaging is a crucial problem. We demonstrated a deep-learning-based power-enhancement (PE) model in a harmonic generation microscope (HGM), including second harmonic generation (SHG) and third harmonic generation (THG). Our model can predict high-power HGM images from low-power images, greatly reducing the risk of phototoxicity and photodamage. Furthermore, the PE model trained only on normal skin data can also be used to predict abnormal skin data, enabling the dermatopathologist to successfully identify and label cancer cells. The PE model shows potential for in-vivo and ex-vivo HGM imaging.

Discontinuity third harmonic generation microscopy for label-free imaging and quantification of intraepidermal nerve fibers.

Label-free imaging methodologies for nerve fibers rely on spatial signal continuity to identify fibers and fail to image free intraepidermal nerve endings (FINEs). Here, we present an imaging methodology-called discontinuity third harmonic generation (THG) microscopy (dTHGM)-that detects three-dimensional discontinuities in THG signals as the contrast. We describe the mechanism and design of dTHGM and apply it to reveal the bead-string characteristics of unmyelinated FINEs. We confirmed the label-free capability of dTHGM through a comparison study with the PGP9.5 immunohistochemical staining slides and a longitudinal spared nerve injury study. An intraepidermal nerve fiber (IENF) index based on a discontinuous-dot-connecting algorithm was developed to facilitate clinical applications of dTHGM. A preliminary clinical study confirmed that the IENF index was highly correlated with skin-biopsy-based IENF density (Pearson's correlation coefficient R = 0.98) and could achieve differential identification of small-fiber neuropathy (p = 0.0102) in patients with diabetic peripheral neuropathy.

In vivo metabolic imaging of insulin with multiphoton fluorescence of human insulin-Au nanodots.

Functional human insulin-Au nanodots (NDs) are synthesized for the in vivo imaging of insulin metabolism. Benefiting from its efficient red to near infrared fluorescence, deep tissue subcellular uptake of insulin-Au NDs can be clearly resolved through a least-invasive harmonic generation and two-photon fluorescence (TPF) microscope. In vivo investigations on mice ear and ex vivo assays on human fat tissues conclude that cells with rich insulin receptors have higher uptake of administrated insulin. Interestingly, the insulin-Au NDs can even permeate into lipid droplets (LDs) of adipocytes. Using this newly discovered metabolic phenomenon of insulin, it is found that enlarged adipocytes in type II diabetes mice have higher adjacent/LD concentration contrast with small-sized ones in wild type mice. For human clinical samples, the epicardial adipocytes of patients with diabetes and coronary artery disease (CAD) also show elevated adjacent/LD concentration contrast. As a result, human insulin-Au nanodots provide a new approach to explore subcellular insulin metabolism in model animals or patients with metabolic or cardiovascular diseases.

High-depth-resolution 3-dimensional radar-imaging system based on a few-cycle W-band photonic millimeter-wave pulse generator.

We demonstrate that a near-single-cycle photonic millimeter-wave short-pulse generator at W-band is capable to provide high spatial resolution three-dimensional (3-D) radar imaging. A preliminary study indicates that 3-D radar images with a state-of-the-art ranging resolution of around 1.2 cm at the W-band can be achieved.