Raman microscopy for skin evaluation.

Many dermatological studies have had limited success in revealing skin function because conventional histological methods are known to affect skin components. Recent progress in non-invasive optical imaging has enabled non-invasive visualization of the structure of each skin layer. However, it remains difficult to identify individual skin components. Alternatively, it is possible to obtain molecular vibrational signatures using spontaneous Raman scattering microscopy. Spontaneous Raman scattering microscopy requires long acquisition times and is rarely applied to skin imaging, especially because skin components, such as water and transepidermal agents, undergo relatively rapid changes. Consequently, non-linear Raman microscopies, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, have gradually been applied to acquire molecular imaging of skin tissue. In this review, the applications of Raman microscopies used to evaluate skin and research trends are presented. The applications of spontaneous Raman microscopy to in vivo human skin evaluation are first demonstrated with typical applications. Finally, the latest application of coherent Raman scattering microscopy to visualize 3D intracellular morphologies in the human epidermis during differentiation is described.

Visualizing intra-medulla lipids in human hair using ultra-multiplex CARS, SHG, and THG microscopy.

We performed label-free imaging of human-hair medulla using multi-modal nonlinear optical microscopy. Intra-medulla lipids (IMLs) were clearly visualized by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging. Two groups of IMLs were found: second harmonic generation (SHG) active and inactive. By combining SHG analysis with CARS, the two groups were identified as free fatty acids and wax esters, respectively.

Changes in facial moisture distribution and feelings of moisture/dryness among various environmental temperatures and humidities in summer and winter.

BACKGROUND: As environmental conditions vary depending on area of residence, consideration of environmental temperature and humidity conditions is crucial for detection of actual skin conditions in daily life. In this study, we determined changes in facial moisture and sensory evaluation distributions in various environmental temperature and humidity conditions. MATERIALS AND METHODS: An original near-infrared (NIR) imaging system was used to obtain moisture distributions. Sensory evaluations of feelings of moisture/dryness were graded, and changes were compared among 10 healthy Japanese female subjects in four different environmental temperature and humidity conditions (28°C, 60% RH; 28°C, 20% RH; 15°C, 60% RH; 15°C, 20% RH) in summer and winter. RESULTS: Skin moisture was lower at high temperatures and higher at low temperatures. Feelings of dryness on bare skin were high in low humidity. Sensitivity of feelings of moisture and dryness was high around the center of the cheekbones between side of the eyes and the mouth, but the same was not true of the moisture distribution. Moisture level was lower in winter at high temperatures, especially under the eyes near the side of the nose ridge, while the sense of dryness was not strong. These divergences between sensory evaluation and moisture level indicate the presence of a "hidden dry situation." CONCLUSION: Changes in moisture level and sensory evaluation scores in facial skin varied among environmental conditions, which differed between summer and winter, even under the same environmental temperature and humidity.

Changes of choroidal structure and circulation after water drinking test in normal eyes.

PURPOSE: To determine whether water drinking test (WDT) alters choroidal structure using binarization of enhanced depth imaging optical coherence tomographic (EDI-OCT) images. METHODS: We performed a prospective study of 57 eyes of 57 normal subjects. The intraocular pressure (IOP), EDI-OCT images, and laser speckle flowgraphic images were recorded at baseline, and at 15, 30, 45, and 120 min after the WDT. The EDI-OCT images were converted to binary images using ImageJ software, and we examined luminal area, interstitial area, whole choroidal area, the ratio of luminal area to whole choroidal area (L/W ratio), subfoveal choroidal thickness (SCT), and central retinal thickness (CRT). RESULTS: The luminal area, L/W ratio, whole choroidal area, and IOP were significantly increased 30 min after water drinking; levels returned to the baseline at 120 min. No significant changes were found in the CRT and interstitial area. The fluctuations in the SCT after water intake were significantly correlated with those in the L/W ratio and luminal area but not with those of the interstitial area. The choroidal blood flow velocity was significantly decreased at 30 min. Fluctuations in the luminal area, L/W ratio, and whole choroidal area were significantly correlated with IOP fluctuations. CONCLUSIONS: The changes in the SCT after water drinking were mainly due to the changes in the choroidal vascular space. Dilatations of the choroidal vessels after water drinking may lead to choroidal thickening and subsequent IOP elevation. These findings should be considered in the evaluation of choroidal structure in patients with retinal disease.

CORRELATIONS BETWEEN CHOROIDAL STRUCTURES AND VISUAL FUNCTIONS IN EYES WITH RETINITIS PIGMENTOSA.

PURPOSE: To investigate the choroidal structures in the enhanced depth imaging optical coherence tomographic images in eyes with retinitis pigmentosa (RP) and to determine correlations between the choroidal structures and visual functions. METHODS: The enhanced depth imaging optical coherence tomographic images of 100 eyes with typical RP and 60 age-, sex-, and axial length-matched normal eyes were binarized using ImageJ. The cross-sectional luminal and stromal areas of the inner and outer subfoveal choroid of 1,500-µm width were measured. The inner choroid included the choriocapillaris and medium vessel layer, and the outer choroid included the larger vessel layer. RESULTS: In the inner choroid, the luminal area and the ratio of luminal/total choroidal area (L/C ratio) were significantly smaller in RP than in controls (P = 0.010, P < 0.001, respectively), whereas the stromal area was not significantly different (P = 0.114). The inner choroidal L/C ratio was significantly correlated with the best-corrected visual acuity, mean deviation, foveal sensitivity, width of the ellipsoid zone, and central foveal thickness in RP after adjusting for the axial length, age, and sex (all P < 0.005). CONCLUSION: The significant correlations between the inner choroidal structures and the visual functions and retinal structures indicate that the choroidal structures are altered in association with the progression of RP.

Changes of choroidal structure after treatment for primary intraocular lymphoma: retrospective, observational case series.

BACKGROUND: We report changes of choroidal structure determined by binarization of enhanced depth imaging optical coherence tomographic (EDI-OCT) images after treatment for primary intraocular lymphoma (PIOL). METHODS: Five eyes of four patients with PIOL were examined by EDI-OCT before and 6 months after intravitreal methotrexate injections. In addition, 15 eyes of 15 normal individuals controlled by age and refractive error were examined by EDI-OCT. Binarization of the EDI-OCT images was performed using publicly accessible software (ImageJ). The examined area of the subfoveal choroid was 1,500 µm wide, and the dark areas that represented the luminal areas were traced by the Niblack method. Wilcoxon signed rank test was used to determine the significance of changes in the subfoveal choroidal thickness, interstitial area, and luminal area. Mann-Whitney U test was used to compare the parameters in the eyes with pretreatment PIOL and normal control eyes. RESULTS: The subfoveal choroidal thickness was significantly decreased after treatment (P = 0.0431). In the binarized images, the interstitial area was significantly decreased after treatment (P = 0.0431), while the luminal area was not significantly changed (P = 0.8927). After delayed onset of PIOL, increased interstitial area, thickened choroid and unchanged luminal area were observed in one eye. The interstitial area and choroidal thickness were significantly increased in the eyes with pretreatment PIOL compared with the normal control eyes (P = 0.0207, P = 0.0495, respectively), while the luminal area was not significantly different (P = 0.2752). CONCLUSIONS: After treatment for PIOL, the EDI-OCT images showed a thinner choroid, and binarization of the EDI-OCT images showed significantly decreased interstitial areas compared with the luminal areas. The binarized EDI-OCT images can provide useful information on choroidal structure in eyes with PIOL, and combining these images with intraocular interleukin levels or fundus autofluorescence images should provide valuable information for determining the PIOL activity.

Binarization of enhanced depth imaging optical coherence tomographic images of an eye with Wyburn-Mason syndrome: a case report.

BACKGROUND: To report a thicker choroid and larger choroidal luminal area in an eye with Wyburn-Mason syndrome. To the best of our knowledge, this is the first report demonstrating an increase in the choroidal thickness and the luminal area in a case of Wyburn-Mason syndrome. In addition, we report the changing appearance of retinal arteriovenous malformations over a 16-year period. CASE PRESENTATION: A 27-year-old woman, who was diagnosed with Wyburn-Mason syndrome at age 11 years, visited our clinic. Her best-corrected visual acuity was 20/12.5 in the right eye and light perception in the left eye. Severely dilated, tortuous vascular loops were distributed from the optic disc over all four quadrants of the left fundus. The vascular loops in some areas were more dilated and tortuous than 16 years earlier. Optical coherence tomography (OCT) showed retinal edema with cystic changes and enlarged choroidal vessel lumens in the left eye. The subfoveal choroidal thickness was manually measured by the caliper function in the enhanced depth imaging OCT (EDI-OCT) images. Binarization of the EDI-OCT images was performed with publicly accessible ImageJ software. The examined area of the subfoveal choroid was 1,500 µm wide, and the dark areas representing the luminal areas were traced by the Niblack method. After determining the distance of each pixel, the luminal area was automatically calculated. The subfoveal choroidal thickness was 250 µm in the right eye and 462 µm in the left eye. The luminal area of the 1,500-µm-wide subfoveal choroid was computed to be 307,165.6 µm(2) in the right eye and 545,780.7 µm(2) in the left eye. CONCLUSIONS: The EDI-OCT images showed a thicker choroid, and binarization of the EDI-OCT images showed that the luminal areas were significantly larger in the affected eye, suggesting a dilatation of the choroidal vessels. The results demonstrated that conversion of EDI-OCT images to binary images was a useful method to quantify the choroidal structure.

Near-infrared imaging of water in human hair.

BACKGROUND/PURPOSE: The water content of hair can be evaluated by weighing, the Karl Fischer method, and from electrical properties. However, these methods cannot be used to study the distribution of water in the hair. Imaging techniques are required for this purpose. In this study, a highly sensitive near-infrared (NIR) imaging system was developed for evaluating water in human hair. The results obtained from NIR imaging and conventional methods were compared. METHODS: An extended indium-gallium-arsenide NIR camera (detection range: 1100-2200 nm) and diffuse illumination unit developed in our laboratory were used to obtain a NIR image of hair. A water image was obtained using a 1950-nm interference filter and polarization filter. Changes in the hair water content with relative humidity (20-95% RH) and after immersion in a 7% (w/w) sorbitol solution were measured using the NIR camera and an insulation resistance tester. The changes in the water content after treatment with two types of commercially available shampoo were also measured using the NIR camera. RESULTS: As the water content increased with changes in the relative humidity, the brightness of the water image decreased and the insulation resistance decreased. The brightness in the NIR image of hair treated with sorbitol solution was lower than that in the image of hair treated with water. This shows the sorbitol-treated hair contains more water than water-treated hair. The sorbitol-treated hair had a lower resistance after treatment than before, which also shows that sorbitol treatment increases the water content. With this system, we could detect a difference in the moisturizing effect between two commercially available shampoos. CONCLUSION: The highly sensitive imaging system could be used to study water in human hair. Changes in the water content of hair depended on the relative humidity and treatment with moisturizer. The results obtained using the NIR imaging system were similar to those obtained using a conventional method. Our system could detect differences in the moisturizing effects of two commercially available shampoos.

Alterations in choroidal vascular structures due to serum levels of vascular endothelial growth factor in patients with POEMS syndrome.

A higher serum vascular endothelial growth factor (VEGF) level can cause choroidal thickening in the choroid of patients with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome. We aimed to determine whether fluctuations in serum VEGF levels affect choroidal vascular structures in patients with POEMS syndrome. This retrospective observational case series examined 17 left eyes of 17 patients with POEMS syndrome. Enhanced depth imaging optical coherence tomography (EDI-OCT) images were obtained, and serum VEGF levels were measured at baseline and 6 months after transplantation with dexamethasone (n = 6), thalidomide (n = 8), or lenalidomide (n = 3). EDI-OCT images were binarized using ImageJ software, and we calculated the areas of the whole choroid and the luminal and stromal areas. Subsequently, we determined whether the choroidal vascular structure had changed significantly between baseline and 6 months after treatment. Six months after treatment, serum VEGF levels and the whole choroid, luminal, and stromal areas had decreased significantly compared to the baseline values (all, P < 0.001). The mean luminal area to the whole choroidal area ratio at 6 months after treatment was 0.70 ± 0.03, which was significantly smaller than the ratio at baseline (0.72 ± 0.03; P < 0.001). Whole choroid and luminal area fluctuations were significantly positively correlated with fluctuations in serum VEGF levels (r = 0.626, P = 0.007 and r = 0.585, P = 0.014, respectively). Choroidal thickening induced by VEGF might be caused by increases in the choroidal vessel lumen area. These results may offer insights into the pathogenesis of POEMS syndrome and the role of serum VEGF in choroidal vascular structure, which may apply to other ocular diseases.

Three-Dimensional Analysis of Water Dynamics in Human Skin by Stimulated Raman Scattering.

The stratum corneum (SC), the outermost layer of the skin, has an important function to provide a barrier against dry environments. To evaluate the barrier function and the skin condition, it is crucial to investigate the ability of SC to absorb and retain water. In this study, we demonstrate stimulated Raman scattering (SRS) imaging of three-dimensional SC structure and water distribution when water is absorbed into dried SC sheets. Our results show that the process of water absorption and retention is dependent on the specific sample and can be spatially heterogeneous. We also found that acetone treatment leads to spatially homogeneous retention of water. These results suggest the great potential of SRS imaging in diagnosing skin conditions.

Estimation of Visual Function Using Deep Learning From Ultra-Widefield Fundus Images of Eyes With Retinitis Pigmentosa.

Importance: There is no widespread effective treatment to halt the progression of retinitis pigmentosa. Consequently, adequate assessment and estimation of residual visual function are important clinically. Objective: To examine whether deep learning can accurately estimate the visual function of patients with retinitis pigmentosa by using ultra-widefield fundus images obtained on concurrent visits. Design, Setting, and Participants: Data for this multicenter, retrospective, cross-sectional study were collected between January 1, 2012, and December 31, 2018. This study included 695 consecutive patients with retinitis pigmentosa who were examined at 5 institutions. Each of the 3 types of input images-ultra-widefield pseudocolor images, ultra-widefield fundus autofluorescence images, and both ultra-widefield pseudocolor and fundus autofluorescence images-was paired with 1 of the 31 types of ensemble models constructed from 5 deep learning models (Visual Geometry Group-16, Residual Network-50, InceptionV3, DenseNet121, and EfficientNetB0). We used 848, 212, and 214 images for the training, validation, and testing data, respectively. All data from 1 institution were used for the independent testing data. Data analysis was performed from June 7, 2021, to December 5, 2022. Main Outcomes and Measures: The mean deviation on the Humphrey field analyzer, central retinal sensitivity, and best-corrected visual acuity were estimated. The image type-ensemble model combination that yielded the smallest mean absolute error was defined as the model with the best estimation accuracy. After removal of the bias of including both eyes with the generalized linear mixed model, correlations between the actual values of the testing data and the estimated values by the best accuracy model were examined by calculating standardized regression coefficients and P values. Results: The study included 1274 eyes of 695 patients. A total of 385 patients were female (55.4%), and the mean (SD) age was 53.9 (17.2) years. Among the 3 types of images, the model using ultra-widefield fundus autofluorescence images alone provided the best estimation accuracy for mean deviation, central sensitivity, and visual acuity. Standardized regression coefficients were 0.684 (95% CI, 0.567-0.802) for the mean deviation estimation, 0.697 (95% CI, 0.590-0.804) for the central sensitivity estimation, and 0.309 (95% CI, 0.187-0.430) for the visual acuity estimation (all P < .001). Conclusions and Relevance: Results of this study suggest that the visual function estimation in patients with retinitis pigmentosa from ultra-widefield fundus autofluorescence images using deep learning might help assess disease progression objectively. Findings also suggest that deep learning models might monitor the progression of retinitis pigmentosa efficiently during follow-up.

A Case of Vogt-Koyanagi-Harada Disease-Like Uveitis Induced by Nivolumab and Ipilimumab Combination Therapy.

Nivolumab and ipilimumab are widely used immune checkpoint inhibitors (ICPIs) for the treatment of metastatic melanoma. ICPIs cause an array of side effects called immune-related adverse events (IRAEs) due to activation of an immune response. ICPI-uveitis can cause irreversible vision loss if untreated. There are few reports of recurrent Vogt-Koyanagi-Harada (VKH) disease-like uveitis induced by nivolumab and ipilimumab. We report a case of VKH disease-like uveitis recurrence after resuming ICPIs. A 73-year-old man with advanced melanoma was referred to our clinic with visual loss 25 days after starting nivolumab/ipilimumab. His corrected visual acuity was 0.5 in the right eye and 0.02 in the left eye. Enhanced-depth imaging optical coherence tomography (EDI-OCT) showed marked choroid thickening. The patient was diagnosed with VKH disease-like uveitis due to IRAEs. Subtenon injection of triamcinolone acetonide was performed, and nivolumab/ipilimumab was suspended, but serous retinal detachment (SRD) markedly worsened and choroidal detachment appeared. With 2 courses of steroid pulse therapy and oral steroids, SRD disappeared, and corrected visual acuity recovered in both eyes. Five months after the first injection, exacerbation of melanoma was observed, and nivolumab and oral steroids were restarted. Six weeks later, an increase in choroidal thickness was observed with EDI-OCT and diagnosed as a recurrence of VKH disease-like uveitis. Monitoring for the recurrence of VKH disease-like uveitis during the administration of ICPIs, even after uveitis is treated, is essential. Assessment of choroidal thickness with EDI-OCT may be useful for detecting early signs of VKH disease-like uveitis.

Changes in Choroidal Component Ratio and Circulation After Coffee Intake in Healthy Subjects.

Purpose: The effects of coffee intake on the ratio of stromal and luminal components in the choroid and the underlying mechanism remain unclear. This prospective cross-sectional study aimed to explore how coffee intake affects the choroidal component ratio and circulation. Methods: Forty-nine right eyes of healthy adult volunteers were evaluated as the coffee intake group. Thirty-two right eyes of healthy volunteers served as the control group. The participants consumed 185 mL of coffee or water, respectively, and the systemic hemodynamics, enhanced-depth imaging optical coherence tomographic (EDI-OCT) images, and foveal mean blur rate (MBR), an indicator of blood flow velocity, were recorded at baseline and after coffee or water intake. The EDI-OCT images were binarized using ImageJ software, and subfoveal choroidal thickness (SCT) and whole, luminal, and stromal choroidal areas were calculated. Results: In the coffee intake group, significant decreases in SCT and luminal area peaked at 60 minutes after intake (both P < 0.001), whereas a significant increase in MBR peaked at 30 minutes (P < 0.001). No significant stromal area fluctuations were observed. SCT and luminal area fluctuations exhibited a significant positive correlation (r = 0.978, P < 0.001). Significant negative correlations of luminal area fluctuations with MBR fluctuations were observed by stepwise regression analysis (r = -0.220, P < 0.001). The control group exhibited no significant fluctuations. Conclusions: Coffee-induced choroidal thinning may result mainly from a reduction in the choroidal vessel lumen, and this vessel lumen reduction correlated with an increased choroidal blood flow velocity after coffee intake. These coffee-induced changes in choroidal component ratio and circulation should be considered when evaluating choroids.

Changes in choroidal structure following intravitreal aflibercept therapy for retinal vein occlusion.

AIMS: To examine the choroidal change accompanying retinal vein occlusion (RVO) in detail, we measured changes in choroidal structure after intravitreal aflibercept (IVA) injections for RVO using binarisation of enhanced depth imaging optical coherence tomographic (EDI-OCT) images and assessed associations with clinical outcome. METHODS: Retrospective, observational case series. Forty treatment-naïve patients (10 central, 18 major branch and 12 macular branch RVO) were examined by EDI-OCT before and 1, 3 and 6 months after IVA injections. EDI-OCT images were binarised using ImageJ. Subfoveal cross-sectional areas of the luminal, stromal and total choroid over a 1500 µm span were measured and the stromal area to total choroidal area (S/C) ratio was calculated. RESULTS: Compared to normal contralateral eyes, afflicted eyes at baseline exhibited significantly greater stromal area (p<0.001), total choroidal area (p=0.001) and S/C ratio (p<0.001), but no difference in luminal area (p=0.083). The stromal area, S/C ratio and total choroidal area were significantly reduced in afflicted eyes at 1, 3 and 6 months after IVA (all p<0.006). Baseline S/C ratio was significantly correlated with baseline visual acuity (VA), baseline central retinal thickness (CRT) and VA and CRT improvement at 1, 3 and 6 months post-treatment even after adjusting for the axial length, age and sex (all p<0.012). CONCLUSION: RVO induces substantial oedema of the choroidal stromal area that is detectable by binarisation of EDI-OCT images. This stromal oedema likely stems from high intraocular vascular endothelial growth factor levels. Changes in choroidal structure may be used to assess severity and prognosis of RVO.

The evaluation of the amount of cis- and trans-urocanic acid in the stratum corneum by Raman spectroscopy.

Urocanic acid (UCA) is a major component in the epidermis which absorbs ultraviolet light (UV) and is isomerized from the trans to the cis form upon UV exposure. This study aimed to evaluate the amounts of cis- and trans-UCA in the stratum corneum (SC) in vivo using Raman spectroscopy. UV damage has previously been investigated by calculating the amount of trans-UCA by fitting Raman spectra. cis-UCA at pH 5.0 was also included in the fitting algorithm in this study. For validation, the SC was stripped from the subjects and the levels of cis- and trans-UCA were measured using high performance liquid chromatography (HPLC). Comparison between these two methods was performed at UV exposure and non-UV exposure sites on the volar forearm of 10 healthy Japanese subjects. Finally, the protective capacity of sunscreen was evaluated by Raman spectroscopy on the volar forearm of 18 healthy subjects. The amount of cis-UCA increased and the amount of trans-UCA decreased after UV exposure when measured by Raman spectroscopy. This was confirmed by HPLC analysis of the SC stripped from the same area of skin used in the Raman measurement. The protection against the increase in the cis-UCA and decrease in the trans-UCA amount after UV exposure by the application of sunscreen products was observed using Raman spectroscopy. The results show this method can be used to measure cis- and trans-UCA levels in vivo.

In vivo simultaneous measurement of urea and water in the human stratum corneum by diffuse-reflectance near-infrared spectroscopy.

BACKGROUND/PURPOSE: As near-infrared spectroscopy is a powerful non-invasive technique, information about several functional groups such as OH, NH, and CH can be monitored simultaneously. This study aimed to coincidentally measure urea and water contents in the human stratum corneum after the treatment of urea-containing cream, in vivo, by a Fourier transform near-infrared spectrometer with a fiber-optic probe. METHOD: Diffuse reflectance spectra of heels of five healthy Japanese volunteers in the 1250-2500 nm region were measured in vivo before and after the 2-h treatment of urea-containing cream. The relative urea content of the heel was calculated from the peak height ratio at 1976 and 2175 nm in the second derivative spectra and that of water was obtained from the 1900 nm water band and that at 2175 nm. Water mobility was calculated from the peak top wavelength of the 1900 nm water band. RESULTS: The estimated water content tended to increase 1 and 2 h after the treatment compared with before the treatment and subsequently gradually decreased with time. The estimated urea content significantly increased 1 h after the treatment. Significant changes in peak wavelength to shorter wavelengths 1, 2, and 4 h after the treatment was observed, suggesting that the water mobility increased. CONCLUSION: The estimated urea and water contents in heels before and after the treatment with urea-containing cream were monitored separately in vivo by diffuse reflectance near-infrared spectroscopy. We demonstrated the potential of NIR spectroscopy for evaluating the efficacy of topical application of a urea-containing cream to the skin in vivo.

Changes in the depth profile of water in the stratum corneum treated with water.

BACKGROUND/PURPOSE: Water in the stratum corneum (SC) plays an important role in keeping the skin surface soft and smooth. Information regarding the depth profile of the molecular composition of the skin, such as water and free amino acids, can be obtained non-invasively using the recently developed confocal Raman spectrometer. In this study, we investigated changes in the depth profile of water in the SC treated with water. We also unveil a relationship between the depth profile patterns of water content in the SC and cutaneous sensation. METHODS: Depth profiles of Raman spectra in the region 2600-4000 cm(-1) were obtained using a 671-nm laser at 2 mum intervals from the skin surface toward the interior with a confocal Raman spectrometer. Water content (mass%), expressed in grams of water per 100 g wet tissue, was calculated from the water-to-protein ratio of the Raman band. Skin surface temperature was measured by an infrared irradiation thermometer. Changes in the depth profile of water and skin temperature were measured at 1, 5, and 10 min after the application of water to the skin surface. In addition, questionnaires regarding cutaneous sensation were administered. RESULTS: Water content in the middle to lower part of the SC increased with increased water-application time. Warming of the skin during water application increased the water-penetration amount, depth, and holding time in the SC. Steamer application increased water content particularly in the upper part of the SC. Increasing water content in the upper part of the SC was associated with cutaneous sensations, such as hydrate and water-rich feelings. On the other hand, the increasing water content in the middle and lower part of the SC was associated with water penetration and tension feelings. CONCLUSION: The water content in the upper part of the SC changed easily. When water was applied externally, the water content in both the upper and middle/lower part of the SC increased with increasing water-application time. In addition, warming of the skin during water application increased the water-penetration depth, amount, and holding time of water in the SC. Thus, we were able to control the depth profile of water in the SC by externally applying water. The location of water in the SC also affected cutaneous sensations.

Choroidal Vasculature from Ultra-Widefield Images without Contrast Dye and Its Application to Vogt-Koyanagi-Harada Disease.

PURPOSE: To develop a method to obtain ultra-widefield choroidal vessel images with a fundus camera without using dye, and its application in Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Experimental study and case series. PARTICIPANTS: Patients with unilateral retinal disorders and those with VKH disease who had undergone ultra-widefield imaging were studied. METHODS: Indocyanine green angiography (ICGA) and the 635-nm wavelength Optos ultra-widefield fundus photography (Optos, United Kingdom) images (Optos635-nm) were processed by KagoEye 3 software. The 2 types of images were overlapped. The degree of overlapped blood vessel areas of the ICGA image was taken as the matching ratio. The matching ratio was obtained for the peripheral, posterior pole, and overall areas. In addition, changes in the fundus findings were followed up with Optos635-nm image analysis in patients with VKH disease. The degree of visibility of the choroidal vessels was evaluated for 5 stages. The clarity scores and the longitudinal findings were compared. MAIN OUTCOME MEASURES: The matching ratios between the ICGA images and the Optos635-nm images processed by KagoEye3 software were determined. RESULTS: Initially, 10 healthy eyes were studied. The matching ratios for the overall area, the peripheral area, and the posterior pole area of the ICGA image and the Optos635-nm image were 64.09%, 74%, and 63.10%, respectively. The correlations between the choroidal blood vessel matching ratio and the ocular axial length and refractive error were not significant, but the matching ratio was correlated significantly with the age. The average clarity score in 12 VKH disease patients was 1.6 ± 0.85 before treatment, which was significantly improved to 4.2 ± 0.75 after 1 month (P < 0.05). Many hyporeflective spotty lesions were observed on the Optos635-nm images, which coincided with hyperfluorescent dots on the ICGA images. The lesions gradually disappeared and the vortex vein became visible after treatment. CONCLUSIONS: The ultra-widefield Optos635-nm images processed by KagoEye3 software can exaggerate images of the choroidal vessels in widefield fundus images without using dye. Because this method is noninvasive, it is applicable to a variety of diseased and healthy eyes.

Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation.

Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.