Objective monitoring of laser tattoo removal in human volunteers using an innovative optical technique: A proof of principle.

OBJECTIVES: Assess the suitability of the technique for objective monitoring of laser tattoo removal by an extended treatment protocol. MATERIALS AND METHODS: One half of the tattoo in the first volunteer was treated with nanosecond and the other half with picosecond laser pulses at 1064 nm. In the second subject, four test areas were treated repeatedly using different radiant exposures from 1.5 to 6 J/cm2 . Measurements of diffuse reflectance spectra and photothermal radiometric transients were performed 4-20 weeks after each treatment session. Inverse Monte Carlo analysis based on a three-layer model of tattooed skin was applied to assess the tattoo characteristics and analyze their changes. RESULTS: The results clearly indicate a gradual reduction of the ink content and an increase of the subsurface depth of the tattoo layer with all treatments at a radiant exposure of 3 J/cm2 or higher. The observed dependences on laser pulse duration, radiant exposure, and a number of treatments are in excellent agreement with visual fading of the tattoo. CONCLUSIONS: The presented methodology enables noninvasive characterization of tattoos in human skin and objective monitoring of the laser removal treatment.

Influence of crystal structure and oxygen vacancies on optical properties of nanostructured multi-stoichiometric tungsten suboxides.

Four distinct tungsten suboxide (WO3-x) nanomaterials were synthesized via chemical vapour transport reaction and the role of their crystal structures on the optical properties was studied. These materials grow either as thin, quasi-2D crystals with the WnO3n-1formula (in shape of platelets or nanotiles), or as nanowires (W5O14, W18O49). For the quasi-2D materials, the appearance of defect states gives rise to two indirect absorption edges. One is assigned to the regular bandgap occurring between the valence and the conduction band, while the second is a defect-induced band. While the bandgap values of platelets and nanotiles are in the upper range of the reported values for the suboxides, the nanowires' bandgaps are lower due to the higher number of free charge carriers. Both types of nanowires sustain localized surface plasmon resonances, as evidenced from the extinction measurements, whereas the quasi-2D materials exhibit excitonic transitions. All four materials have photoluminescence emission peaks in the UV region. The interplay of the crystal structure, oxygen vacancies and shape can result in changes in optical behaviour, and the understanding of these effects could enable intentional tuning of selected properties.

Noninvasive Monitoring of Dynamical Processes in Bruised Human Skin Using Diffuse Reflectance Spectroscopy and Pulsed Photothermal Radiometry.

We have augmented a recently introduced method for noninvasive analysis of skin structure and composition and applied it to monitoring of dynamical processes in traumatic bruises. The approach combines diffuse reflectance spectroscopy in visible spectral range and pulsed photothermal radiometry. Data from both techniques are analyzed simultaneously using a numerical model of light and heat transport in a four-layer model of human skin. Compared to the earlier presented approach, the newly introduced elements include two additional chromophores (ß-carotene and bilirubin), individually adjusted thickness of the papillary dermal layer, and analysis of the bruised site using baseline values assessed from intact skin in its vicinity. Analyses of traumatic bruises in three volunteers over a period of 16 days clearly indicate a gradual, yet substantial increase of the dermal blood content and reduction of its oxygenation level in the first days after injury. This is followed by the emergence of bilirubin and relaxation of all model parameters towards the values characteristic for healthy skin approximately two weeks after the injury. The assessed parameter values and time dependences are consistent with existing literature. Thus, the presented methodology offers a viable approach for objective characterization of the bruise healing process.

Temperature Depth Profiles Induced in Human Skin In Vivo Using Pulsed 975 nm Irradiation.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the temperature depth profiles induced in human skin in vivo by using a pulsed 975 nm diode laser (with 5 ms pulse duration) and compare them with those induced by the more common 532 nm (KTP) and 1,064 nm (Nd:YAG) lasers. Quantitative assessment of the energy deposition characteristics in human skin at 975 nm should help design of safe and effective treatment protocols when using such lasers. STUDY DESIGN/MATERIALS AND METHODS: Temperature depth profiles induced in the human skin by the three lasers were determined using pulsed photothermal radiometry (PPTR). This technique involves time-resolved measurement of mid-infrared emission from the irradiated test site and reconstruction of the laser-induced temperature profiles using an earlier developed optimization algorithm. Measurements were performed on volar sides of the forearms in seven volunteers with healthy skin. At irradiation spot diameters of 3-4 mm, the radiant exposures were 0.24, 0.36, and 5.7 J/cm2 for the 975, 532, and 1,064 nm lasers, respectively. RESULTS: Upon normalization to the same radiant exposure of 1 J/cm 2 , the assessed maximum temperature rise in the epidermis averaged 0.8 °C for the 975 nm laser, 7.4 °C for the 532 nm, and 0.6 °C for the 1,064 nm laser. The characteristic subsurface depth to which 50% of the absorbed laser energy was deposited was on average 0.31 mm at 975 nm irradiation, and slightly deeper at 1,064 nm, and 0.15 mm at 532 nm. The experimentally obtained relations were reproduced in a dedicated numerical simulation. CONCLUSIONS: The assessed energy deposition characteristics show that the pulsed 975 nm diode laser is very suitable for controlled heating of the upper dermis as required, for example, for nonablative skin rejuvenation. The risks of nonselective overheating of the epidermis and subcutis are significantly reduced in comparison with irradiation at 532 and 1,064 nm, respectively. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Noninvasive assessment of skin structure by combined photothermal radiometry and optical spectroscopy: coregistration with multiphoton microscopy.

We are combining two optical techniques, pulsed photothermal radiometry (PPTR) and diffuse reflectance spectroscopy (DRS), for noninvasive assessment of the structure and composition of human skin in vivo. The analysis involves simultaneous multidimensional fitting of the measured PPTR signals and DRS spectra with predictions of a numerical model of light transport (Monte Carlo) in a four-layer model optical model of human skin, accounting for the epidermis, papillary and reticular dermis, and subcutis. The assessed epidermal thickness values were tested by coregistration with a multiphoton microscope, which provides vertical sectioning capability based on two-photon excited fluorescence and second-harmonic generation in selected skin components. The comparison shows that these values correspond well to the maximal epidermal thicknesses measured in the multiphoton microscopy images, the rete ridges.

Optical properties of biomimetic probes engineered from erythrocytes.

Light-activated theranostic materials offer a potential platform for optical imaging and phototherapeutic applications. We have engineered constructs derived from erythrocytes, which can be doped with the FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-mimicking transducers (NETs). Herein, we investigated the effects of changing the NETs mean diameter from micron- (≈4 µm) to nano- (≈90 nm) scale, and the ICG concentration utilized in the fabrication of NETs from 5 to 20 µM on the resulting absorption and scattering characteristics of the NETs. Our approach consisted of integrating sphere-based measurements of light transmittance and reflectance, and subsequent utilization of these measurements in an inverse adding-doubling algorithm to estimate the absorption (µ a) and reduced scattering (µ s') coefficients of these NETs. For a given NETs diameter, values of µ a increased over the approximate spectral band of 630-860 nm with increasing ICG concentration. Micron-sized NETs produced the highest peak value of µ a when using ICG concentrations of 10 and 20 µM, and showed increased values of µ s' as compared to nano-sized NETs. Spectral profiles of µ s' for these NETs showed a trend consistent with Mie scattering behavior for spherical objects. For all NETs investigated, changing the ICG concentration minimally affected the scattering characteristics. A Monte Carlo-based model of light distribution showed that the presence of these NETs enhanced the fluence levels within simulated blood vessels. These results provide important data towards determining the appropriate light dosimetry parameters for an intended light-based biomedical application of NETs.

Dissolution Mechanism of Upconverting AYF4:Yb,Tm (A = Na or K) Nanoparticles in Aqueous Media.

The dissolution of upconverting AYF4:Yb,Tm (A = Na or K) nanoparticles (UCNPs) in aqueous media was systematically studied. UCNPs with a cubic structure and sizes of between 10 and 33 nm were synthesized solvothermally in ethylene glycol at 200 °C. The UCNPs of both compositions showed an upconversion fluorescence emission characteristic of Tm(3+). The effects of the A cation, the particle size, the temperature, the pH, and the composition of the aqueous medium on the dissolution of the UCNPs were evaluated. The degree of dissolution was determined from the fraction of dissolved fluoride (F(-)) using potentiometry. Unexpectedly, the composition of aqueous media had the most significant effect on the dissolution of the UCNPs. The highest degree of dissolution and rate were measured for the phosphate-buffered saline (PBS), which can be explained by the formation of stable lanthanide compounds with phosphates. The degree of dissolution was much lower in water and in the phthalate buffer, which was attributed to the release of F(-) as a result of the hydrolysis of the UCNPs' surfaces.

Correlations of light scattering properties in human skin with the person's age assessed using a non-invasive technique.

We analyze the influence of a person's age on the thicknesses and reduced scattering coefficients of the epidermis and dermis in visible part of the spectrum. Their values were assessed using a non-invasive technique which combines pulsed photothermal radiometry and diffuse reflectance spectroscopy with Monte Carlo modeling of light transport in a four-layer model of skin. The analysis is affected by the strong influences of the melanin content on the reduced scattering coefficient of the epidermis, a epi, and blood content in the case of dermis (a der). Separating their contributions reveals a significant decrease of a der with the person's age at an average rate of -0.25 mm-1 per decade, while the contribution of blood in the papillary dermis amounts to 1.0 mm-1%-1. Meanwhile, no influence of the person's age was found on a epi and the thicknesses of the epidermis or dermis.

Energy deposition profile in human skin upon irradiation with a 1,342 nm Nd:YAP laser.

BACKGROUND AND OBJECTIVES: Nd:YAP laser emitting at 1,342 nm appears promising for nonablative skin rejuvenation treatment, based on favorable absorption properties of water and melanin in this part of the spectrum. A quantitative determination of energy deposition characteristics of Nd:YAP in normal human skin should enable design of a safe and effective treatment protocol for future human studies. STUDY DESIGN: Energy deposition profile of a prototype Nd:YAP laser was determined using pulsed photothermal radiometry. This technique involves time-resolved measurement of mid-infrared emission from a sample after pulsed laser irradiation. The laser-induced temperature depth profile is reconstructed from the radiometric transients using a custom optimization algorithm, developed and tested earlier in our group. Measurements were performed on the extremities of four healthy volunteers at low radiant exposure (2.8 J/cm(2) ). For the purpose of comparison, energy deposition characteristics of commercial Nd:YAG and KTP lasers (at 1,064 and 532 nm, respectively), were also determined at the same test sites. RESULTS: On average, the Nd:YAP laser deposits 50% of the absorbed energy within the top 0.36 mm of skin and 90% within 0.86 mm, which is significantly shallower than the Nd:YAG laser. The ratio between the dermal versus epidermal heating is more favorable and shows a smaller inter- and intra-patient variance as compared to both Nd:YAG and KTP laser. CONCLUSIONS: Energy deposition characteristics of the 1,342 nm Nd:YAP laser are very suitable for controlled heating of the upper dermis, as required for nonablative skin rejuvenation. The risks of overheating the epidermis or subcutis should be significantly reduced in comparison with the 1,064 nm Nd:YAG laser.

Locally induced shockwaves for selective perforation of cargo loaded lipid vesicles with temporal and spatial control.

Controlled poration of lipid membranes is crucial for numerous biomimetic applications such as targeted drug delivery. Although several chemical and physical mechanisms have been proposed for the poration of synthetic membranes, achieving good temporal and spatial control remains a challenge. In this study, we introduce a novel method for membrane poration that utilizes the mechanical shockwave generated by the photo-acoustic effect, which occurs when an optically opaque microparticle is illuminated by a near-infrared laser of optical tweezers. We show that the shockwave effectively porates membranes of giant unilamellar vesicles in close proximity to the microparticle without damaging nearby cells, which is a desirable outcome for potential targeted drug delivery. The poration effect is nonspecific and operates on both liquid and gel phase membranes. Since the photo-acoustic effect can be triggered by standard optical tweezers, this method holds broad applicability in various experimental settings within the field of soft matter research.

Photocoagulation of dermal blood vessels with multiple laser pulses in an in vivo microvascular model.

BACKGROUND/OBJECTIVES: Current laser therapy of port wine stain (PWS) birthmarks with a single laser pulse (SLP) does not produce complete lesion removal in the majority of patients. To improve PWS therapeutic efficacy, we evaluated the performance of an approach based on multiple laser pulses (MLP) to enhance blood vessel photocoagulation. STUDY DESIGN: The hamster dorsal window chamber model was used. Radiant exposure (RE), pulse repetition rate (f(r)), total number of pulses (n(p)), and length of vessel irradiated were varied. Blood vessels in the window were irradiated with either SLP with RE of 4-7 J/cm(2) or MLP with RE per pulse of 1.4-5.0 J/cm(2), f(r) of 0.5-26.0 Hz, and n(p) of 2-5. The laser wavelength was 532 nm and pulse duration was 1 ms. Either a 2 mm vessel segment or entire vessel branch was irradiated. Digital photographs and laser speckle images of the window were recorded before and at specific time points after laser irradiation to monitor laser-induced blood vessel structural and functional changes, respectively. RESULTS: We found that: (1) for a SLP approach, the RE required to induce blood vessel photocoagulation was 7 J/cm(2) as compared to only 2 J/cm(2) per pulse for the MLP approach; (2) for MLP, two pulses at a repetition rate of 5 Hz and a RE of 3 J/cm(2) can induce photocoagulation of more than 80% of irradiated blood vessel; and (3) irradiation of a longer segment of blood vessel resulted in lower reperfusion rate. CONCLUSIONS: The MLP approach can induce blood vessel photocoagulation at much lower RE per pulse as compared to SLP. The 5 Hz f(r) and the need for two pulses are achievable with modern laser technology, which makes the MLP approach practical in the clinical management of PWS birthmarks.

Optimisation of Amphiphilic-Polymer Coatings for Improved Chemical Stability of NaYF4-based Upconverting Nanoparticles.

NaYF4 nanoparticles codoped with Yb3+ and Tm3+ exhibit upconversion fluorescence in near-infrared and visible spectral range. Consequently, such upconverting nanoparticles (UCNPs) can be used as contrast agents in medical diagnostics and bioassays. However, they are not chemically stable in aqueous dispersions, especially in phosphate solutions. Protective amphiphilic-polymer coatings based on poly(maleic anhydride-alt-octadec-1-ene) (PMAO) and bis(hexamethylene)triamine (BHMT) were optimised to improve the chemical stability of UCNPs under simulated physiological conditions. Morphologies of the bare and coated UCNPs was inspected with transmission electron microscopy. All samples showed intense UC fluorescence at ~800 nm, typical for Tm3+. The colloidal stability of aqueous dispersions of bare and coated UCNPs was assessed by dynamic light scattering and measurements of zeta potential. The dissolution of UCNP in phosphate-buffered saline at 37 °C, was assessed potentiometrically by measuring the concentration of the dissolved fluoride. Protection against the dissolution of UCNPs was achieved by PMAO and PMAO crosslinked with BHMT.

Deep tissue localization and sensing using optical microcavity probes.

Optical microcavities and microlasers were recently introduced as probes inside living cells and tissues. Their main advantages are spectrally narrow emission lines and high sensitivity to the environment. Despite numerous novel methods for optical imaging in strongly scattering biological tissues, imaging at single-cell resolution beyond the ballistic light transport regime remains very challenging. Here, we show that optical microcavity probes embedded inside cells enable three-dimensional localization and tracking of individual cells over extended time periods, as well as sensing of their environment, at depths well beyond the light transport length. This is achieved by utilizing unique spectral features of the whispering-gallery modes, which are unaffected by tissue scattering, absorption, and autofluorescence. In addition, microcavities can be functionalized for simultaneous sensing of various parameters, such as temperature or pH value, which extends their versatility beyond the capabilities of standard fluorescent labels.

Numerical optimization of sequential cryogen spray cooling and laser irradiation for improved therapy of port wine stain.

BACKGROUND AND OBJECTIVE: Despite application of cryogen spray (CS) precooling, customary treatment of port wine stain (PWS) birthmarks with a single laser pulse does not result in complete lesion blanching for a majority of patients. One obvious reason is nonselective absorption by epidermal melanin, which limits the maximal safe radiant exposure. Another possible reason for treatment failure is screening of laser light within large PWS vessels, which prevents uniform heating of the entire vessel lumen. Our aim is to identify the parameters of sequential CS cooling and laser irradiation that will allow optimal photocoagulation of various PWS blood vessels with minimal risk of epidermal thermal damage. STUDY DESIGN AND METHODS: Light and heat transport in laser treatment of PWS are simulated using a custom 3D Monte Carlo model and 2D finite element method, respectively. Protein denaturation in blood and skin are calculated using the Arrhenius kinetic model with tissue-specific coefficients. Simulated PWS vessels with diameters of 30-150 µm are located at depths of 200-600 µm, and shading by nearby vessels is accounted for according to PWS histology data from the literature. For moderately pigmented and dark skin phototypes, PWS blood vessel coagulation and epidermal thermal damage are assessed for various parameters of sequential CS cooling and 532-nm laser irradiation, i.e. the number of pulses in a sequence (1-5), repetition rate (7-30 Hz), and radiant exposure. RESULTS: Simulations of PWS treatment in darker skin phototypes indicate specific cooling/irradiation sequences that provide significantly higher efficacy and safety as compared to the customary single-pulse approach across a wide range of PWS blood vessel diameters and depths. The optimal sequences involve three to five laser pulses at repetition rates of 10-15 Hz. CONCLUSIONS: Application of the identified cooling/irradiation sequences may offer improved therapeutic outcome for patients with resistant PWS, especially in darker skin phototypes.

NaYF4-based upconverting nanoparticles with optimized phosphonate coatings for chemical stability and viability of human endothelial cells.

The increasing interest in upconverting nanoparticles (UCNPs) in biodiagnostics and therapy fuels the development of biocompatible UCNPs platforms. UCNPs are typically nanocrystallites of rare-earth fluorides codoped with Yb3+and Er3+or Tm3+. The most studied UCNPs are based on NaYF4but are not chemically stable in water. They dissolve significantly in the presence of phosphates. To prevent any adverse effects on the UCNPs induced by cellular phosphates, the surfaces of UCNPs must be made chemically inert and stable by suitable coatings. We studied the effect of various phosphonate coatings on chemical stability andin vitrocytotoxicity of the Yb3+,Er3+-codoped NaYF4UCNPs in human endothelial cells obtained from cellular line Ea.hy926. Cell viability of endothelial cells was determined using the resazurin-based assay after the short-term (15 min), and long-term (24 h and 48 h) incubations with UCNPs dispersed in cell-culture medium. The coatings were obtained from tertaphosphonic acid (EDTMP), sodium alendronate and poly(ethylene glycol)-neridronate. Regardless of the coating conditions, 1 - 2 nm-thick amorphous surface layers were observed on the UCNPs with transmission electron microscopy. The upconversion fluorescence was measured in the dispersions of all UCNPs. Surafce quenching in aqueous suspensions of the UCNPs was reduced by the coatings. The dissolution degree of the UCNPs was determined from the concentration of dissolved fluoride measured with ion-selective electrode after the ageing of UCNPs in water, physiological buffer (i.e., phosphate-buffered saline-PBS) and cell-culture medium. The phosphonate coatings prepared at 80 °C significantly suppressed the dissolution of UCNPs in PBS while only minor dissolution of bare and coated UCNPs was measured in water and cell-culture medium. The viability of human endothelial cells was significantly reduced when incubated with UCNPs, but it increased with the improved chemical stability of UCNPs by the phosphonate coatings with negligible cytotoxicity when coated with EDTMP at 80 °C.

Toward a Flexible and Efficient TiO2 Photocatalyst Immobilized on a Titanium Foil.

Titanium foils of different thicknesses were anodized, and the photocatalytic activity of the resulting TiO2 nanotube (NT) layers was determined. All of the titanium foils were anodized simultaneously under identical experimental conditions to avoid the influence of the aging of the anodizing electrolyte and other anodization parameters, such as voltage, time, and temperature. To characterize the microstructures of the titanium foils, we used electron backscatter diffraction (EBSD), scanning electron microscopy (SEM), and stylus profilometry analyses. The adhesion was tested with a Scotch tape test and the morphology of the TiO2 NTs was studied in detail using the SEM technique, while the surface areas of the TiO2 NTs were determined using a three-dimensional (3D) optical interference profilometer. With X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), the chemical composition and structure of TiO2 oxide were established. The degradation of caffeine under UV irradiation was measured with a high-precision UV-vis-IR spectrophotometer, and the photoluminescence method was used to confirm the photocatalytic behavior of the TiO2 NT layers. The influence of the intrinsic properties, including twinning and the grain boundaries of the starting titanium foils with similar chemical compositions, was determined and explained. Finally, we identified the main characteristics that define a highly effective and flexible photocatalyst.

Formation of phosphonate coatings for improved chemical stability of upconverting nanoparticles under physiological conditions.

Upconverting nanoparticles (UCNPs) are being extensively investigated for applications in bioimaging because of their ability to emit ultraviolet, visible, and near-infrared light. NaYF4 is one of the most suitable host matrices for producing high-intensity upconversion fluorescence; however, UCNPs based on NaYF4 are not chemically stable in aqueous media. To prevent dissolution, their surfaces should be modified. We studied the formation of protective phosphonate coatings made of ethylenediamine(tetramethylenephosphonic acid), alendronic acid, and poly(ethylene glycol)-neridronate on cubic NaYF4 nanoparticles and hexagonal Yb3+,Er3+-doped upconverting NaYF4 nanoparticles (ß-UCNPs). The effects of synthesis temperature and ultrasonic agitation on the quality of the coatings were studied. The formation of the coatings was investigated by transmission electron microscopy, zeta-potential measurements, and infrared spectroscopy. The quality of the phosphonate coatings was examined with respect to preventing the dissolution of the NPs in phosphate-buffered saline (PBS). The dissolution tests were carried out under physiological conditions (37 °C and pH 7.4) for 3 days and were followed by measurements of the dissolved fluoride with an ion-selective electrode. We found that the protection of the phosphonate coatings can be significantly increased by synthesizing them at 80 °C. At the same time, the coatings obtained at this temperature suppressed the surface quenching of the upconversion fluorescence in ß-UCNPs.

Predictive model for the quantitative analysis of human skin using photothermal radiometry and diffuse reflectance spectroscopy.

We have recently introduced a novel methodology for the noninvasive analysis of the structure and composition of human skin in vivo. The approach combines pulsed photothermal radiometry (PPTR), involving time-resolved measurements of mid-infrared emission after irradiation with a millisecond light pulse, and diffuse reflectance spectroscopy (DRS) in the visible part of the spectrum. Simultaneous fitting of both data sets with respective predictions from a numerical model of light transport in human skin enables the assessment of the contents of skin chromophores (melanin, oxy-, and deoxy-hemoglobin), as well as scattering properties and thicknesses of the epidermis and dermis. However, the involved iterative optimization of 14 skin model parameters using a numerical forward model (i.e., inverse Monte Carlo - IMC) is computationally very expensive. In order to overcome this drawback, we have constructed a very fast predictive model (PM) based on machine learning. The PM involves random forests, trained on ∼9,000 examples computed using our forward MC model. We show that the performance of such a PM is very satisfying, both in objective testing using cross-validation and in direct comparisons with the IMC procedure. We also present a hybrid approach (HA), which combines the speed of the PM with versatility of the IMC procedure. Compared with the latter, the HA improves both the accuracy and robustness of the inverse analysis, while significantly reducing the computation times.

A spectrally composite reconstruction approach for improved resolution of pulsed photothermal temperature profiling in water-based samples.

We report on the first experimental evaluation of pulsed photothermal radiometry (PPTR) using a spectrally composite kernel matrix in signal analysis. Numerical studies have indicated that this approach could enable PPTR temperature profiling in watery tissues with better accuracy and stability as compared to the customary monochromatic approximation. By using an optimized experimental set-up and image reconstruction code (involving a projected nu-method and adaptive regularization), we demonstrate accurate localization of thin absorbing layers in agar tissue phantoms with pronounced spectral variation of a mid-infrared absorption coefficient. Moreover, the widths of reconstructed temperature peaks reach 14-17% of their depth, significantly less than in earlier reports on PPTR depth profiling in watery tissues. Experimental results are replicated by a detailed numerical simulation, which enables analysis of the broadening effect as a function of temperature profile amplitude and depth.

Physiological and structural characterization of human skin in vivo using combined photothermal radiometry and diffuse reflectance spectroscopy.

In this proof-of-concept study we combine two optical techniques to enable assessment of structure and composition of human skin in vivo: Pulsed photothermal radiometry (PPTR), which involves measurements of transient dynamics in mid-infrared emission from sample surface after exposure to a light pulse, and diffuse reflectance spectroscopy (DRS) in visible part of the spectrum. The analysis involves simultaneous fitting of measured PPTR signals and DRS with corresponding predictions of a Monte Carlo model of light-tissue interaction. By using a four-layer optical model of skin we obtain a good match between the experimental and model data when scattering properties of the epidermis and dermis are also optimized on an individual basis. The assessed parameter values correlate well with literature data and demonstrate the expected trends in controlled tests involving temporary obstruction of peripheral blood circulation using a pressure cuff, and acute as well as seasonal sun tanning.