In vivo THz imaging of human skin: Accounting for occlusion effects

In vivo terahertz (THz) imaging of human skin needs to be done in reflection geometry due to the high attenuation of THz light by water in the skin. To aid the measurement procedure, there is typically an imaging window onto which the patient places the area of interest. The window enables better pulse alignment and helps keep the patient correctly positioned during the measurement. In this paper, we demonstrate how the occlusion caused by the skin contact with the imaging window during the measurement affects the THz response. By studying both rapid point measurements and imaging over an area of a human volar forearm, we find that even 5 seconds of occlusion affects the THz response. As the occlusion time increases, the skin surface water content increases, resulting in the reduction of the amplitude of the reflected THz pulse, especially in the first 3 minutes. Furthermore, it was found that the refractive index of the volar forearm increased by 10% to 15% after 20 minutes of occlusion. In this work, we examine and propose a model for the occlusion effects due to the quartz window with a view to compensating for its influence.      

In vivo terahertz reflection imaging of human scars during and after the healing process

We use terahertz imaging to measure four human skin scars in vivo. Clear contrast between the refractive index of the scar and surrounding tissue was observed for all of the scars, despite some being difficult to see with the naked eye. Additionally, we monitored the healing process of a hypertrophic scar. We found that the contrast in the absorption coefficient became less prominent after a few months post‐injury, but that the contrast in the refractive index was still significant even months post‐injury. Our results demonstrate the capability of terahertz imaging to quantitatively measure subtle changes in skin properties and this may be useful for improving scar treatment and management.

     

In vivo sensing of rabbit cornea by terahertz technology

A Novel scalable approach using Terahertz (THz) waves together with the electromagnetic field simulation was applied to investigate four rabbits of eight rabbit corneas in vivo. One eye of each rabbits’ corneas was edema induced; the other eye of the corneas served as the control. The simulation revealed the propagation of THz waves at a certain distance along the sub-surface of the cornea. THz spectra have been collected close to the corneal surface by deviating the direct reflection of the THz beam for the edema cornea, the reflected wave intensity for edema corneas is generally larger compared with the control cornea. Upon edema becomes severe at the end of the observation, the reflected wave intensities obtained by detector corresponding to the corneal deep stroma layer approach to the same value for all observed corneas. Good correlation is observed between central corneal thickness measurements and THz wave reflection signal intensities. Our results demonstrated that THz spectroscopy technique could obtain the information from different corneal sublayers.     

Imaging of ex vivo nonmelanoma skin cancers in the optical and terahertz spectral regions Optical and Terahertz skin cancers imaging

We tested the hypothesis that polarization sensitive optical and terahertz imaging may be combined for accurate nonmelanoma skin cancer (NMSC) delineation. Nine NMSC specimens were imaged. 513 μm and 440 nm wavelengths were used for terahertz and optical imaging, respectively. Histopathology was processed for evaluation. Terahertz reflectance of NMSC was quantified. Our results demonstrate that cross‐polarized terahertz images correctly identified location of the tumours, whereas cross‐polarized and polarization difference optical images accurately presented morphological features. Cross‐polarized terahertz images exhibited lower reflectivity values in cancer as compared to normal tissue. Combination of optical and terahertz imaging shows promise for intraoperative delineation of NMSC (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vivo sensing of cutaneous edema: A comparative study of diffuse reflectance,Raman spectroscopy and multispectral imaging

Quantitative noninvasive assessment of water content in tissues is important for biomedicine. Optical spectroscopy is potentially capable of solving this problem; however, its applicability for clinical diagnostics remains questionable. The presented study compares diffuse reflectance spectroscopy, Raman spectroscopy and multispectral imaging in the characterization of cutaneous edema. The source-detector geometries for each method are selected based on Monte Carlo simulations results to detect the signal from the dermis. Then, the kinetics of the edema development is studied for two models. All methods demonstrate synchronous trends for histamine-induced edema: The water content reaches a maximum of 1 hour after histamine application and then gradually decreases. For the venous occlusion, a 51% increase in water content is observed with Raman spectroscopy. The differences in water content estimation by three methods are explained based on the light propagation model. The obtained results are essential for introducing quantitative optical water measurement technology to the clinics.     

In vivo assessment of human skeletal muscle mitochondria respiration in health and disease

One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (³¹P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by ³¹P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests ha ve been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning. (Mol Cell Biochem 174: 11–15, 1997)     

In vivo multiphoton microscopy of melasma

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non‐invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non‐invasive imaging tool for characterizing melasma.     

In vivo imaging the motility of monocyte/macrophage during inflammation in diabetic mice

Diabetes, as a chronic metabolic disease, can impair the immune function of monocytes/macrophages (MMs). However, it is unclear how MM immune response to inflammation with the development of diabetes, and whether immune response around the inflammatory foci depends on the depth in tissue. Footpad provides a classical physiological site for monitoring cellular behavior during inflammation, but limited to the superficial dermis due to the strong scattering of footpad. Herein, we used confocal microscopy to monitor the motility of MMs in deeper tissue around inflammatory foci with the development of type 1 diabetic (T1D) mice through a switchable footpad skin optical clearing window. Delayed‐type hypersensitivity (DTH) model was elicited on the footpad of T1D. Results demonstrated that progressive T1D led to the gradually potentiated MM recruitment and increased expression of monocyte chemoattractant protein‐1 during DTH, but MM migration displacement, motion velocity and motility coefficient were significantly attenuated. Besides, MMs from the deeper dermis had a much larger migration displacement than those from superficial dermis at early stages of DTH but an opposite tendency at late stages for non‐T1D, while progressive T1D obscured this difference gradually. This study will be helpful for investigating the influences of progressive metabolic diseases on immune response. MM motion trajectory at depth of superficial dermis and the deeper dermis at AOVA (heat‐aggregated ovalbumin)—4 hours and AOVA—72 hours on non‐T1D (A) and T1D—4 weeks (B). Mean motility coefficient (C) at the 2 depths. Data were pooled from 6 mice per group. *P < .05 and **P < .01 compared among different T1D disease durations. #P < .05 compared between different depths.      

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vitro and in vivo confocal Raman study of human skin hydration: assessment of a new moisturizing agent, pMPC

The hydration capacities of a biomimetic polymer, 2-methacryloyloxethylphosphorylcholine polymer (pMPC), alone and microencapsulated, in association with another well known hydrating polymer, Hyaluronic acid, were investigated in vitro on skin models and in vivo on volunteers by using confocal Raman microspectroscopy. The hydration impact and the relative water content in the Stratum corneum were calculated from the Raman spectra using the OH (water)/CH3 (protein) ratio. Moreover, the follow-up of the presence of pMPC through the Stratum corneum was possible with confocal Raman microspectroscopy, using a characteristic vibration of pMPC, different from that of the encapsulating material. From our in vitro measurements, the improved hydration of the Stratum corneum was confirmed by the use of the encapsulated form of pMPC, which was higher when combined with Hyaluronic acid. On the basis of these in vitro findings, we validated this trend in in vivo measurements on 26 volunteers, and found a good correlation with the in vitro results. Mechanical and ultrastructural studies have been carried out to demonstrate the positive effects of the pMPC on the Stratum corneum function, namely the interaction with lamellar lipids and the plasticizing effects, which are both supposed to spell out the moisturizing effect. This study demonstrates the efficiency of a original hydrating agent, pMPC, entrapped with Hyaluronic acid in a new type of microcapsules by the use of a novel tool developed for both in vitro and in vivo approaches. This indicates a new step to evaluate and improve new moisturizers in response to the cosmetics or dermatologic demands.     

Optoacoustic monitoring of water content in tissue phantoms and human skin

Skin water content monitoring is important for diagnostics and management of edema, dehydration, and other skin conditions as well as for cosmetic applications. Because optoacoustic (OA) technique has high (optical) contrast and (ultrasound) resolution and significant probing depth, it may be suitable for accurate, noninvasive water content monitoring in the skin. In this work we studied OA response from skin tissue phantoms and human wrist skin in the wavelength range from 1370 nm to 1650 nm using a novel, tunable OPO OA system. We identified optimal wavelengths for OA water content monitoring in different skin layers. The results of our study suggest that the OA technique may become a valuable, quantitative tool for accurate, high-resolution water content monitoring in the skin and other tissues and may find wide applications in dermatology, cosmetology, and tissue trauma management.     

尼罗红示踪纳米制剂在荷瘤裸鼠体内活体成像的应用

目的拟将尼罗红包裹入纳米制剂中,利用其荧光特性来考察纳米制剂在荷瘤鼠体内的肿瘤靶向性与组织分布情况。方法采用CCK-8法检测尼罗红纳米乳[NRNE(O)]对H1688的细胞毒性。建立BALB/c裸鼠肺癌模型,灌胃给予尼罗红混悬液(NRS)及NRNE(O),通过小动物活体成像系统观察其在荷瘤裸鼠中荧光强度的动态分布情况。结果 NRNE(O)具有较低的细胞毒性和良好的生物相容性,并且在体内的吸收量及肿瘤部位的蓄积量均高于NRS;NR在纳米制剂中能快速、稳定、清晰地反映纳米制剂在小鼠体内的动态分布情况。结论本研究中制备的NRNE(O)的稳定性好,为尼罗红作为示踪剂应用于纳米制剂在动物体内的研究提供了重要的研究手段。     

p16INK4A is a robust in vivo biomarker of cellular aging in human skin

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.     

Raman confocal microscopy and biophysics multiparametric characterization of the skin barrier evolution with age

Skin aging is a multifactorial phenomenon that involves alterations at the molecular, cellular and tissue levels. Our aim was to carry out a multiparametric biophysical and Raman characterization of skin barrier between individuals of different age groups (<24 and >70 years old). Our results showed a significant decrease of lipids to proteins ratio overall the thickness of the stratum corneum and higher lateral packing in the outer part of the SC for elderly. This can explain the decrease in trans epidermal water loss measured values rather than only SC thickening. Both age groups showed similar water content at SC surface while elderly presented higher water content in deep SC and viable epidermis. Mechanical measurements showed a decrease in the elasticity and an increase in the fatigability with age and were correlated with partially bound water. Highest correlation and anti-correlation values were observed for the deepest part of the SC and the viable epidermis.     

In vivo detection of human cutaneous beta‐carotene using computational optical clearing

The content of dermal beta‐carotene can be a good indicator showing the body health. Because, it is involved in production of vitamin A maintaining healthy skin and mucous membranes. Also, it reduces the risk of cardiovascular diseases and its antioxidant capacity prevents the formation of cancerous cells. In this work, we use Raman spectroscopy and a low‐cost diffuse reflectance spectroscopy (DRS) to detect the dermal beta‐carotene spectra. We apply computational optical clearing (OC) method to in vivo evaluation the concentration of this chromophore. The results show that Raman spectroscopy is a good tool for in vitro detection of carotenoids but is not able to clearly discriminate the individual carotenoids in skin tissue in vivo. The results also show that using OC enhances the ability of low‐cost diffuse reflection spectroscopy for in vivo detection of dermal beta‐carotene in humans. This method can be used as a low‐cost and portable device to screening the concentration of chromophores such as melanin and carotenoid molecules for oncological studies.     

利用非侵入性光学成像技术监测在体肿瘤的生长

利用电穿孔技术将gfp表达质粒转染于SP2／0细胞,通过G418筛选获得稳定表达GFP的小鼠骨髓瘤细胞株SP2／0—GFP细胞。将稳定的转化细胞移植于同系BALB／c小鼠后腿、耳部皮下及腹腔成瘤,每隔一天对小鼠肿瘤成像,跟踪肿瘤的生长过程。结果表明,探测到的GFP荧光强度和发光范围与肿瘤的生长或消亡相关,从而证明可以利用GFP作为肿瘤细胞的标记,结合光学成像技术,对肿瘤生长过程实时追踪。     

In vivo non-contact measurement of human iris elasticity by optical coherence elastography

Quantifying the mechanical properties of the iris can offer valuable insights into the pathophysiology of primary angle closure glaucoma. However, current techniques for iris elastography remain ex vivo with limited clinical applications. This article describes a proposition for a non-contact and non-invasive air-puff optical coherence elastography (OCE) system that can evaluate iris elasticity in vivo. Ten eyes recruited from seven subjects underwent OCE imaging acquisition under three different illumination conditions. The Young's modulus of each eye was detected and shown to be inversely proportional to the iris length, indicating a relationship between mechanical properties and morphology of the iris. With its noninvasive and high-resolution features, this air-puff system shows great potential for applications in clinical ophthalmology.     

In vivo monitoring of rashes caused by systemic lupus erythematosus disease using snapshot spectral imaging

Monitoring the activity of systemic lupus erythematosus (SLE) is important to patient management. Blood biochemical indexes are commonly assessed but are both time demanding and traumatizing. In this study, a noninvasive and real‐time spatial‐spectral data tool was used to monitor SLE patients through rash spectral data. To build the relationship between the rash spectrum changes and changes in the patients' status, a snapshot hyperspectral Fourier transform imaging spectrometer was built to monitor the rash reflectance changes of hospitalized SLE patients. A simple rash activity index (RAI) which was normally distributed with the doctor's visual rating of rash severity was calculated from hyperspectral images. The sensitivity of the change in RAI is higher than complement 3/4 levels. RAI and anti‐dsDNA antibodies both decreased as the patients recovered. Anti‐dsDNA and complement 3/4 were important indicators of SLE activity suggesting that the RAI directly correlated with patient status. The snapshot spectrometer therefore provides an auxiliary method to monitor SLE disease.