Validation model for Raman based skin carotenoid detection

Raman spectroscopy holds promise as a rapid objective non-invasive optical method for the detection of carotenoid compounds in human tissue in vivo. Carotenoids are of interest due to their functions as antioxidants and/or optical absorbers of phototoxic light at deep blue and near UV wavelengths. In the macular region of the human retina, carotenoids may prevent or delay the onset of age-related tissue degeneration. In human skin, they may help prevent premature skin aging, and are possibly involved in the prevention of certain skin cancers. Furthermore, since carotenoids exist in high concentrations in a wide variety of fruits and vegetables, and are routinely taken up by the human body through the diet, skin carotenoid levels may serve as an objective biomarker for fruit and vegetable intake. Before the Raman method can be accepted as a widespread optical alternative for carotenoid measurements, direct validation studies are needed to compare it with the gold standard of high performance liquid chromatography. This is because the tissue Raman response is in general accompanied by a host of other optical processes which have to be taken into account. In skin, the most prominent is strongly diffusive, non-Raman scattering, leading to relatively shallow light penetration of the blue/green excitation light required for resonant Raman detection of carotenoids. Also, sizable light attenuation exists due to the combined absorption from collagen, porphyrin, hemoglobin, and melanin chromophores, and additional fluorescence is generated by collagen and porphyrins. In this study, we investigate for the first time the direct correlation of in vivo skin tissue carotenoid Raman measurements with subsequent chromatography derived carotenoid concentrations. As tissue site we use heel skin, in which the stratum corneum layer thickness exceeds the light penetration depth, which is free of optically confounding chromophores, which can be easily optically accessed for in vivo RRS measurement, and which can be easily removed for subsequent biochemical measurements. Excellent correlation (coefficient R = 0.95) is obtained for this tissue site which could serve as a model site for scaled up future validation studies of large populations. The obtained results provide proof that resonance Raman spectroscopy is a valid non-invasive objective methodology for the quantitative assessment of carotenoid antioxidants in human skin in vivo.     

Cover Picture: J. Biophotonics 7/2012

The cover illustrates the measurement of human skin carotenoid levels based on pressure mediated reflection spectroscopy. Carotenoids have a widespread distribution in fruits and vegetables, are taken up through the diet, and play an important role in tissue health through their function as antioxidants. Their characteristic absorption in the blue/green spectral range can be quantified in human tissue such as the thumb and tracked upon dietary supplementation. (Picture: I. V. Ermakov and W. Gellermann, pp. 442–453, in this issue)     

Spectral correction for handheld optoacoustic imaging by means of near‐infrared optical tomography in reflection mode

In vivo imaging of tissue/vasculature oxygen saturation levels is of prime interest in many clinical applications. To this end, the feasibility of combining two distinct and complementary imaging modalities is investigated: optoacoustics (OA) and near‐infrared optical tomography (NIROT), both operating noninvasively in reflection mode. Experiments were conducted on two optically heterogeneous phantoms mimicking tissue before and after the occurrence of a perturbation. OA imaging was used to resolve submillimetric vessel‐like optical absorbers at depths up to 25 mm, but with a spectral distortion in the OA signals. NIROT measurements were utilized to image perturbations in the background and to estimate the light fluence inside the phantoms at the wavelength pair (760 nm, 830 nm). This enabled the spectral correction of the vessel‐like absorbers' OA signals: the error in the ratio of the absorption coefficient at 830 nm to that at 760 nm was reduced from 60%‐150% to 10%‐20%. The results suggest that oxygen saturation (SO ₂) levels in arteries can be determined with <10% error and furthermore, that relative changes in vessels' SO ₂ can be monitored with even better accuracy. The outcome relies on a proper identification of the OA signals emanating from the studied vessels.      

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.     

Spectrophotometric measurements of human tissues for the detection of subjacent blood vessels in an endonasal endoscopic surgical approach

Thin slices of human tissues are characterized concerning reflection and transmission in a wavelength range from 400 to 1700 nm. The results are primarily useful to find a wavelength for the detection of subjacent blood vessels during surgical procedures, especially neurological surgery. The measurements have been conducted using a customized measuring station, utilizing two halogen bulb lamps and two spectrometers. This paper focuses on creating a data base with the optical properties of artery, brain, bone, nasal mucosa, and nerve. The spectral distributions are compared among each other, similarities and differences are pointed out. Each tissue has got unique spectral characteristics, whereas typical absorption bands can be found in the overall tissues, especially hemoglobin and water absorption bands. The reflectivity maxima are typically located in the red or near‐infrared. All the transmission maxima are located between 1075 nm and 1100 nm. The measurements have been conducted at the Institute of Anatomy at the University of Leipzig. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Multi‐wavelength light emitting diode‐based disposable optrode array for in vivo optogenetic modulation

We present a light emitting diode (LED)‐based optical waveguide array that can optogenetically modulate genetically targeted neurons in the brain. The reusable part of the system consists of control electronics and conventional multi‐wavelength LED. The disposable part comprises optical fibers assembled with microlens array fabricated on a silicon die. Both parts can be easily assembled and separated by snap fit structure. Measured light intensity is 3.35 mW/mm² at 469 nm and 0.29 mW/mm² at 590 nm when the applied current is 80 mA. In all the tested conditions, the light‐induced temperature rise is under 0.5°C and over 90% of the relative light intensity is maintained at 2 mm‐distance from the fiber tips. We further tested the efficiency of the optical array in vivo at 469 nm. When the optical array delivers light stimulation on to the visual cortex of a mouse expressing channelrhodopsin‐2, the neural activity is significantly increased. The light‐driven neural activity is successfully transformed into a percept of the mouse, showing significant learning of the task detecting the cortical stimulation. Our results demonstrate that the proposed optical array interfaces well with the neural circuits in vivo and the system is applicable to guide animal behaviors.      

2‐D mapping of skin chromophores in the spectral range 500 – 700 nm

The multi‐spectral imaging technique has been used for distant mapping of in‐vivo skin chromophores by analyzing spectral data at each reflected image pixel and constructing 2‐D maps of the relative concentrations of oxy‐/deoxy‐haemoglobin and melanin. Instead of using a broad visible‐NIR spectral range, this study focuses on narrowed spectral band 500–700 nm, speeding‐up the signal processing procedure. Regression analysis confirmed that superposition of three Gaussians is optimal analytic approximation for the oxy‐haemoglobin absorption tabular spectrum in this spectral band, while superposition of two Gaussians fits well for deoxy‐haemoglobin absorption and exponential function – for melanin absorption. The proposed approach was clinically tested for three types of in‐vivo skin provocations: ultraviolet irradiance, chemical reaction with vinegar essence and finger arterial occlusion. Spectral range 500–700 nm provided better sensitivity to oxy‐haemoglobin changes and higher response stability to melanin than two reduced ranges 500–600 nm and 530–620 nm. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of green and red light in lipid accumulation and transcriptional profile of genes implicated in lipid biosynthesis in Chlamydomonas reinhardtii

Microalgae have the potential to accumulate triacylglycerols under different light spectra. In this work, Chlamydomonas reinhardtii was grown under white (400–700 nm), red (650 nm), and green (550 nm) lights. According to our results, red light (650 nm) has a positive effect in the microalgae growth and chlorophyll concentration. About the lipid content, the control culture (white light‐illuminated) reached a 4.4% of dry cell weight (dcw), whereas the culture grown at 550 nm showed an increase of 1.35‐fold in the lipids accumulation (5.96% dcw). Interestingly, the most significant accumulation was found in the culture grown at 650 nm (14.78% dcw) which means 3.36‐fold higher with respect to the white light‐illuminated culture. The most abundant fatty acids found in lipid extracts obtained from the cultures under different light wavelength were palmitic (C16: 0), oleic (C18: 1n9), stearidonic (C18: 4), and linoleic (C18: 2), which are useful in the biodiesel production. Changes in gene expression in response to different wavelength illuminations were assessed; however, an in‐depth analysis of a larger number of genes involved in lipid biosynthesis is necessary to fully explain the highest accumulation of lipids in the culture grown under red light. This approach will be useful to find a sustainable source of lipids for biodiesel production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1404–1411, 2016     

The potential utility of carotenoid‐based coloration as a biomonitor of environmental change

In the past 30 years, carotenoid‐based animal signals have been an intense focus of research because they can potentially broadcast an honest reflection of individual reproductive potential. Our understanding of the underpinning physiological functions of carotenoid compounds is still emerging, however. Here, we argue that wildlife researchers and managers interested in assessing the impact of environmental quality on animal populations should be taking advantage of the signalling function of carotenoid‐based morphological traits. Using birds as a model taxonomic group, we build our argument by first reviewing the strong evidence that the expression of avian carotenoid displays provides an integrated measure of a multitude of diet‐ and health‐related parameters. We then present evidence that human‐induced rapid environmental change (HIREC) impacts the expression of carotenoid signals across different critical periods of a bird’s lifetime. Finally, we argue that variation in signal expression across individuals, populations and species could be quantified relatively easily at a global scale by incorporating such measurements into widespread bird ringing activities. Monitoring the expression of carotenoid‐based coloration could help to identify how the environmental factors linked to HIREC can affect avian populations and allow for potentially detrimental effects on biodiversity to be detected prior to demographic change.     

Temperature regime of biological tissue under photodynamic therapy

An analytical model is proposed to calculate heating of human skin cover under laser light action of photodynamic therapy. A photosensitizer of «Fotolon» is taken as an example. Temperatures of skin surface and of deep dermis regions are studied as a function of time under pulsed and stationary irradiation of skin surface at the wavelength of 665 nm corresponding to the maximum of the photosensitizer absorption band. It is shown that, under the action of a short light pulse, the photosensitizer can lead to an essential temperature rise of dermis due to a considerable increase in its absorption coefficient. However, this rise does not destruct tissue cells because of the short action. Under stationary irradiation, the photosensitizer concentration has a low effect on the temperature regime of tissue. This is related with the specific features in heating of the medium by red light, where the main thermal process in skin is heat transfer over tissue volume from epidermis having a substantially larger absorption coefficient than that of dermis in the said spectral range. The role of blood perfusion in dermis and its effect on the temperature regime of tissue are evaluated.     

Validity and reliability of Raman spectroscopy for carotenoid assessment in cattle skin

Carotenoids are powerful antioxidants capable of helping to protect the skin from the damaging effects of exposure to sun by reducing the free radicals in skin produced by exposure to ultraviolet radiation, and they may also have a physical protective effect in human skin. Since carotenoids are lipophilic molecules which can be ingested with the diet, they can accumulate in significant quantities in the skin. Several studies on humans have been conducted to evaluate the protective function of carotenoids against various diseases, but there is very limited published information available to understand the mechanism of carotenoid bioavailability in animals. The current study was conducted to investigate the skin carotenoid level (SCL) in two cattle skin sets – weaners with an unknown feeding regime and New Generation Beef (NGB) cattle with monitored feed at three different ages. Rapid analytical and sensitive Raman spectroscopy has been shown to be of interest as a powerful technique for the detection of carotenoids in cattle skin due to the strong resonance enhancement with 532 nm laser excitation. The spectral difference of both types of skin were measured and quantified using univariate and linear discriminant analysis. SCL was higher in NGB cattle than weaners and there is a perfect classification accuracy between weaners and NGB cattle skin using carotenoid markers as a basis. Further work carried out on carotenoid rich NGB cattle skin of 8, 12 and 24 months of age identified an increasing trend in SCL with age. The present work validated the ability of Raman spectroscopy to determine the skin carotenoid level in cattle by comparing it with established HPLC methods. There is an excellent correlation of R² = 0.96 between the two methods that could serve as a model for future application for larger population studies.     

用于胃癌检测的漫反射光谱

胃癌正严重地威胁人类的健康,为了开发一种可以诊断早期胃癌的新型光学检测技术,建立了一套简便的组织体漫反射光谱检测装置.首先,根据组织体的光学特性,介绍光谱检测方法的基本原理.然后,利用组织癌变导致的组织体光学特性变化,建立漫反射光谱检测的实验装置.最后,利用该实验装置分别获取正常胃组织和胃癌组织的漫反射光谱.实验结果表明:癌变和正常胃组织的漫反射光谱在可见光区域有明显差别,特别是在波长为500 nm和630 nm处,但是漫反射光谱检测无法分辨来自组织不同层的光谱信息.这些结果说明漫反射光谱检测装置可用于胃癌的辅助检测.     

Application of a sensitive near‐field microwave microprobe to the nondestructive characterization of microbial rhodopsin

We study the opto‐electrical properties of Natronomonas pharaonis sensory rhodopsin II (NpSRII) by using a near‐field microwave microprobe (NFMM) under external light illumination. To investigate the possibility of application of NFMM to biological macromolecules, we used time dependent properties of NPSRII before/after light activation which has three distinct states – ground‐state, M‐state, and O‐state. The diagnostic ability of NFMM is demonstrated by measuring the microwave reflection coefficient (S₁₁) spectrum of NpSRII under steady‐state illumination in the wavelength range of 350–650 nm. Moreover, we present microwave reflection coefficient S₁₁ spectra in the same wavelength range for two fast‐photocycling rhodopsins: green light‐absorbing proteorhodopsin (GPR) and Gloeobacter rhodopsin (GR). In addition the frequency sweep shift can be detected completely even for tiny amounts of sample (～10^–3 OD of rhodopsin). Based on these results NFMM shows both very high sensitivity for detecting conformational changes and produces a good time‐resolved spectrum. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differences in visible light‐induced pigmentation according to wavelengths: a clinical and histological study in comparison with UVB exposure

The visible light spectrum is wide, and it can be hypothesized that all the wavelengths between 400–700 nm do not induce the same photobiological effects on pigmentation. We assessed the potential pro‐pigmenting effects of two single wavelengths located at both extremities of the visible spectrum: the blue/violet line (λ = 415 nm) and the red line (λ = 630 nm). We made colorimetric, clinical, and histological assessments with increasing doses of those lights on healthy volunteers. Then, we compared these irradiations to non‐exposed and UVB‐exposed skin. Colorimetric and clinical assessments showed a clear dose effect with the 415‐nm irradiation, in both skin type III and IV subjects, whereas the 630 nm did not induce hyperpigmentation. When compared to UVB irradiation, the blue–violet light induced a significantly more pronounced hyperpigmentation that lasted up to 3 months. Histological examination showed a significant increase of keratinocyte necrosis and p53 with UVB, as compared to 415‐ and 630‐nm exposures.     

MICROENVIRONMENTAL CONTROL OF PHOTOSYNTHESIS AND PHOTOSYNTHESIS-COUPLED RESPIRATION IN AN EPILITHIC CYANOBACTERIAL BIOFILM1

The photosynthetic performance of an epilithic cyano-bacterial biofilm was studied in relation to the in situ light field by the use of combined microsensor measurements of O₂, photosynthesis, and spectral scalar irradiance. The high density of the dominant filamentous cyanobacteria (Oscillatoria sp.) embedded in a matrix of exopolymers and bacteria resulted in a photic zone of < 0.7 mm. At the biofilm surface, the prevailing irradiance and spectral composition were significantly different from the incident light. Multiple scattering led to an intensity maximum for photic light (400–700 nm) of ca. 120% of incident quantum irradiance at the biofilm surface. At the bottom of the euphotic zone in the biofilm, light was attenuated strongly to < 5–10% of the incident surface irradiance. Strong spectral signals from chlorophyll a (440 and 675 nm) and phycobilins (phycoerythrin 540–570 nm, phycocyanin 615–625 nm) were observed as distinct maxima in the scalar irradiance attenuation spectra in the upper 0.0–0.5 mm of the biofilm. The action spectrum for photosynthesis in the cyanobacterial layer revealed peak photosynthetic activity at absorption wavelengths of phycobilins, whereas only low photosynthesis rates were induced by light absorption of carotenoids (450–550 nm). Respiration rates in light- and dark-incubated biofilms were determined using simple flux calculations on measured O₂ concentration profiles and photosynthetic rates. A significantly higher areal O₂ consumption was found in illuminated biofilms than in dark-incubated biofilms. Although photorespiration accounted for part of the increase, the enhanced areal O₂ consumption of illuminated biofilms could also be ascribed to a deeper oxygen penetration in light as well as an enhanced volumetric O₂ respiration in and below the photic zone. Gross photosynthesis was largely unaffected by increasing flow velocities, whereas the O₂ flux out of the photic zone, that is, net photosynthesis, increased with flow velocity. Consequently, the amount of produced O₂ consumed within the biofilm decreased with increasing flow velocity. Our data indicated a close coupling of photosynthesis and respiration in biofilms, where the dissolved inorganic carbon requirement of the photo-synthetic population may largely be covered by the respiration of closely associated populations of heterotrophic bacteria consuming a significant part of the photosynthetically produced oxygen and organic carbon.     

Spectral kinetic modeling and long‐term behavior assessment of Arthrospira platensis growth in photobioreactor under red (620 nm) light illumination

The ability to cultivate the cyanobacterium Arhtrospira platensis in artificially lightened photobioreactors using high energetic efficiency (quasi‐monochromatic) red LED was investigated. To reach the same maximal productivities as with the polychromatic lightening control conditions (red + blue, P/2e^? = 1.275), the need to work with an optimal range of wavelength around 620 nm was first established on batch and continuous cultures. The long‐term physiological and kinetic behavior was then verified in a continuous photobioreactor illuminated only with red (620 nm) LED, showing that the maximum productivities can be maintained over 30 residence times with only minor changes in the pigment content of the cells corresponding to a well‐known adaptation mechanism of the photosystems, but without any effect on growth and stoichiometry. For both poly and monochromatic incident light inputs, a predictive spectral knowledge model was proposed and validated for the first time, allowing the calculation of the kinetics and stoichiometry observed in any photobioreactor cultivating A. platensis, or other cyanobacteria if the parameters were updated. It is shown that the photon flux (with a specified wavelength) must be used instead of light energy flux as a relevant control variable for the growth. The experimental and theoretical results obtained in this study demonstrate that it is possible to save the energy consumed by the lightening device of photobioreactors using red LED, the spectral range of which is defined according to the action spectrum of photosynthesis. This appears to be crucial information for applications in which the energy must be rationalized, as it is the case for life support systems in closed environments like a permanent spatial base or a submarine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

Temperature regime of biological tissue under photodynamic therapy

An analytical model is proposed to calculate heating of human skin cover under laser light action of photodynamic therapy. A photosensitizer of "Fotolon" is taken as an example. Temperatures of skin surface and of deep dermis regions are studied as a function of time under pulsed and stationary irradiation of skin surface at the wavelength of 665 nm corresponding to the maximum of the photosensitizer absorption band. It is shown that, under the action of a short light pulse, the photosensitizer can lead to an essential temperature rise of dermis due to a considerable increase in its absorption coefficient. However, this rise does not destruct tissue cells because of the short action. Under stationary irradiation, the photosensitizer concentration has a low effect on the temperature regime of tissue. This is related with the specific features in heating of the medium by red light, where the main thermal process in skin is heat transfer over tissue volume from epidermis having a substantially larger absorption coefficient than that of dermis in the said spectral range. The role of blood perfusion in dermis and its effect on the temperature regime of tissue are evaluated.