Optical Characteristics Of The Skin With Dark Circles Using Multiphoton Microscopy

BackgroundDark circles under the eyes are common and affect the appearance of all ages with varying degrees.Dark circles are thought to be associated with sleep deprivation,smoking,and life stress,and symptoms tend to decrease as these factors decrease,although these links have not been proven.In human skin,the distribution and content of melanin in the epidermis and dermis determine the basic color of the skin,and the proportion and structure of dermal collagen-elastin determine the thickness,light transmittance,and aging state of the skin.Previous studies believed that the types of dark circles mainly included dark pigmentation type with relatively increased melanocytes and melanin granules,and purple shadow type with thin skin resulting in exposure of subcutaneous blood vessels and orbicularis oculi muscle,as well as their mixed types.Due to the lack of quantitative analysis of the skin components of dark circles,the diagnostic classification standards and severity of dark circles are vague,and the specific parameters of its treatment are not clear enough.For melanin and collagen and elastin markers,traditional staining methods require biopsy and dye operation of specimens,which are invasive and time-consuming.Although the application of confocal reflectance microscopy and Visa-CR devices has achieved some success,such methods lack sensitivity and specificity for melanin granules.Therefore,in the quantitative analysis of melanin granules and collagen-elastin in dark circles’ skin,a non-invasive,accurate,rapid,and label-free method is very necessary.In recent years,with the rapid development of medical engineering,Multiphoton Microscopy(MPM),as a non-invasive and non-labeled imaging tool,represents a new frontier of biomedical research.ObjectiveIn this study,pump-probe imaging was used to image melanin granules in the skin tissue samples of the lower eyelid,and the second harmonic generation(SHG)and twophoton excited fluorescence(TPEF)were used to image collagen and elastin fibers in the dermis.Through the quantitative analysis and parameter statistics of the skin components of dark circles,non-invasive diagnostic classification and quantification of severity were carried out to achieve the purpose of accurate diagnosis and treatment of dark circles.Materials and Methods1.Histological process and pump-probe imaging of lower eyelid skin tissue:Lower eyelid skin tissue samples obtained during lower blepharoplasty were cryopreserved in liquid nitrogen at-196℃.The tissue sections were cut into 6-μm thick sections,and three adjacent slices were selected,one of which was not stained for nonlinear spectroscopy,and the other two were subjected to HE and FeSO4 staining.Samples were collected in strict compliance with the ethical requirements and institutional rules of human clinical research,and informed consent of all patients participating in the study was obtained.A dual-output femtosecond laser system providing two phase-locked femtosecond lasers with an 80 MHz repetition rate.One 120 fs laser with an 850 nm wavelength was used as the probe beam.The other 200 fs laser centered at 1045 nm,which served as the pump beam,was modulated by an acoustic-optic modulator at 2.8 MHz.The two beams were collinearly combined through a dichroic mirror.A 40× water immersion objective was used to focus the light on the sample,and an oil condenser was used to collect the laser from the sample.Two filters(ET845/55 m,ET795/150 m)were used to filter out the pump beam,the probe beam was detected by a photodiode,and the probe beam loss signal was extracted by a lock-in amplifier.2.Statistical analysis of pump-probe images and FeSO4 staining:Pure melanin solution samples were prepared as the reference standard for melanin pump-probe imaging.First,the transient absorption spectra(decay curve)of standard substance and lower eyelid skin samples were obtained respectively and analyzed to confirm that the target region in the pump-probe image of the sample was melanin granules.Then,the melanin content index(MCI)in pump-probe images and FeSO4 staining and the intraclass correlation coefficient(ICC)of the MCIs were calculated respectively;meanwhile,the correlation between MCI value and L*value of the patient’s facial image was analyzed.At last,the mean MCI and MFI(mean fluorescence intensity)of the epidermal basal layer and superficial dermis were calculated respectively.3.SHG and TPEF imaging of the lower eyelid skin:A picosecond tunable laser is used to provide an excitation beam tuned to a wavelength of 800 nm at a repetition rate of 80 MHz.After beam broadening,two-dimensional grating scanning is carried out by a scanning galvanometer system.The sample is then focused with a 60X water-immersion objective and the laser reflected from the sample is collected.Two filters were used to filter out the incident beam,one filter(ET395/25 M-2P)was used for SHG imaging,and the other filter(ET520/60 M-2P)was used for TPEF imaging.The signal beam was detected by electron emission using a photomultiplier tube(PMT)and the data were recorded.4.Data collection and statistical analysis of SHG and TPEF images:SHG and TPEF images were imported into Image J software,and the Second Harmonic to Autofluorescence Aging Index of Dermis(SAAID)of lower eyelid skin was calculated.The Orientation Index of Collagen Bundles(OICB)was calculated after Fast Fourier Translation(FFT)of SHG images.Five fields of the same size were randomly selected to calculate the mean values of SAAID and OICB.Finally,the SAAID and OICB values of different types of dark circles and normal skin,as well as different ages were statistically analyzed to evaluate the changes in the dermis with age and the differences among different types of skin.Results and Analysis1.Facial imaging and histological staining analysis:The skin of the middle,inner and lower eyelids of the purple shadow skin showed purple,and the skin of the upper and lower eyelid of the pigmented skin showed brown and dark pigmentation,and the middle half area was slightly serious.HE staining results were similar to FeSO4 staining results.Melanin granules mainly concentrate in the epidermal basal layer of the lower eyelid skin,and they also disperse in the dermis.The distribution characteristics of purple shadow skin melanin granules are similar to that of the normal lower eyelid skin,but the density of melanin increases slightly.The melanin granules in the dermis of pigmented skin gather into pigment masses.2.Specificity and reliability analysis of pump-probe images for melanin imaging:The melanin in the basal cell layer appears as a layer of bright green light with a typical papillary shape and scattered melanin granules can also be seen in the dermis.Consistent with FeSO4 staining results,pump-probe images showed the same precise detail in the same area.The transient absorption spectra of the skin tissue section of the lower eyelid were consistent with that of the pure melanin solution.Therefore,it was confirmed that the target signal region in the pump-probe image of the experimental sample was indeed melanin granules.The ICC of the MCIs obtained by pump-probe imaging and FeSO4 staining was 0.997(p<0.001),indicating that the pump-probe imaging method is reliable for quantitative analysis of melanin.In addition,the MCI was negatively correlated with the L*value and the linear fitting of the L*value and the MCIs had almost the same slope demonstrating that the pump-probe imaging method had high accuracy for melanin imaging and quantification.3.Quantitative analysis of the skin with different types of dark circles:Both the MCI and the MFI were significantly increased in dark pigmentation skin compared with purple shadow skin and normal skin,and there were statistical differences(p<0.05);the MCI and the MFI of purple shadow skin were also slightly higher than that of the normal skin,but the difference was not statistically significant.In pump-probe images,the MCI value of the epidermal basal layer of pigmentation black eye reached 4.28-6.04%,and the MFI value reached 2628.63-2789.21(AU).At the same time,the MCI value of the superficial dermis was 1.90-3.50%,and the MFI value was 2714.44-2837.52(AU).The S AAID of dark pigmentation skin was significantly lower than that of purple shadow skin,fluctuating between-0.44 and-0.20,indicating that the content proportion of dermal collagen in dark pigmentation skin under the eye was significantly lower than that of purple shadow skin.The OICB of purple shadow skin was significantly higher than that of the normal lower eyelid skin,indicating that the collagen fiber bundles of skin with purple shadow tend to be aligned in a more consistent direction,which is easier to pass through natural light,and making the purple-shaded state of orbicularis oculi muscle and subcutaneous blood vessels be revealed.4.Histological staining and SHG and TPEF image analysis of skin with dark circles of different ages:HE staining results showed that with the increase of age,the irregular papillae of the epidermal basal layer gradually tended to be flat or even completely flat.The number and layer of cells in the epidermal layer decreased gradually,and the thickness also decreased gradually.The fibrous organization of the dermis becomes loose,fine gradually,and appears bigger gap even.SHG signals are dominated by collagens,and TPEF signals are dominated by elastin interweave to form a spatial network architecture.With the aging of patients with dark circles,the SHG signal in the superficial dermis gradually decreased,the structure of collagen bundles gradually became sparse,their volume and density became significantly smaller,and the fluorescence intensity also decreased significantly.Moreover,with the increase of age,the SAAID gradually decreased,while the OICB gradually increased,and there were significant differences among different age groups(p<0.05).This indicates that the content of collagen fibers decreases with aging and damage,and the alignment direction tends to be more consistent.This is because the content of collagen fiber bundles and dermal thickness decrease with age,causing the fiber bundles to compress and become straighter.Conclusion1.In this study,pump-probe imaging technology was used to analyze dark circles skin and normal lower eyelid skin.The results showed that the pump-probe image was consistent with FeSO4 staining,confirming that the method was reliable and accurate in the quantitative analysis of melanin granules.2.In this study,SHG and TPEF techniques were also used for joint imaging of skin with dark circles and normal lower eyelid skin,and it was found that SHG signals dominated by collagen fibers and TPEF signals dominated by elastic fibers were interwoven to form a spatial network architecture.3.Quantitative analysis of skin components in dark circles showed that the MCI and MFI values of pigmentation in dark circles were significantly increased,the S AAID value was significantly lower than that of purple shadow dark circles,and the OICB value of purple shadow dark circles was significantly higher than that of normal lower eyelid skin.4.SHG and TPEF images of skin with dark circles of different ages showed that the content of collagen fiber gradually decreases with the increase of the age of patients with dark circles,but the alignment direction tends to be more consistent.5.A junior clinician can use MPM to categorize dark circles and quantify melanin at different skin levels in noninvasive,nonfixed,and unlabeled conditions to select treatments such as lasers at different treatment depths or fillers at different treatment doses.