第一部分：多光子显微镜与皮肤共聚焦显微镜在健康皮肤的对比

背景：近年来，皮肤成像技术的快速发展实现了对皮肤疾病的实时、活体的检测。多光子显微镜（multiphoton microscopy，MPM）作为一种具有亚微米分辨率的新型影像仪器，能够实现细胞层级的成像。与传统的皮肤反射共聚焦显微镜（reflectance confocal microscopy，RCM）相似，MPM同样能够实现皮肤各层的横断面成。目前MPM在临床应用中的普及程度较低，且两种仪器在皮肤成像中优势尚不明确，缺乏对这两种仪器之间的对比研究。

目的：本研究旨在比较MPM与RCM在皮肤各层的成像优势，评估这两种技术在皮肤厚度测量中的差异，探索MPM在临床中的实际应用价值，为临床皮肤成像提供新的技术选择。

方法：本研究为单中心、横断面研究，于2023年1月至2023年3月期间，招募了56名健康志愿者，由三名医生对身体不同部位（面颊、前臂和背部）进行评估及测量。使用MPM和RCM分别评估了皮肤各层图像（包括角质层、颗粒层、棘层、真表皮交界层和真皮浅层）。此外，对比两种仪器测量不同身体部位的表皮厚度差别。最后，利用MPM可以区分弹力纤维及胶原纤维信号的优势，计算真皮二次谐波与自发荧光老化指数（second harmonic and autofluorescence aging index，SAAID），使用多重线性回归分析了影响SAAID的因素。

结果：MPM在颗粒层、胶原纤维和弹性纤维的观察中更具优势，超过90%的图像评分为“优秀”（p < 0.001），而RCM则在真表皮交界处的观察更具优势，超过90%的图像评分为“优秀”（p < 0.001）。面颊部位的表皮厚度高于前臂和背部，MPM测得的不同部位表皮厚度均低于RCM（RCM中分别为82 ± 8μm、76 ± 9μm、72 ± 9μm，MPM中分别为72 ± 11μm、65 ± 9μm、60 ± 8μm）。40岁以下人群的表皮厚度高于40岁及以上的人群，且表皮厚度随年龄增长减少（p < 0.05）。男性与女性的表皮厚度无明显差异（p > 0.05）。SAAID随着年龄的增长而下降，女性的SAAID下降更为迅速，面颊SAAID下降更为迅速。

结论：MPM作为新型无创诊断技术，对于颗粒层、弹力纤维、胶原纤维的成像更具优势，而RCM在真表皮交界处更具成像优势。两种仪器均能准确测量表皮厚度，且表皮厚度在年龄、性别及不同部位之间存在差异。MPM具有评估皮肤老化程度的优势，这为临床提供新的评价指标。

第二部分：活体多光子显微镜在银屑病中的运用：一种新型疾病特征分析技术

背景：银屑病（psoriasis）是一种慢性、系统性的炎症性疾病，临床表现为覆盖银白色鳞屑的红色斑块。银屑病的诊断和鉴别诊断主要依赖于临床评估及病理活检。然而，肉眼评估较为主观，病理活检可能导致瘢痕或感染。因此，无创光学活检对银屑病诊断和治疗检测具有重要价值。多光子显微镜（multiphoton microscopy，MPM）是一种新型的无创、实时、活体成像技术，可以提供亚微米分辨率，实现单个细胞的结构观察，提供表皮至真皮的分层扫描及三维成像。目前尚无MPM对银屑病的相关研究。

目的：探索MPM在斑块型银屑病中的特征表现及诊断价值，为银屑病的诊断提供新的无创影像手段选择。

方法：本研究为单中心、观察性研究，于2024年2月7日至4月7日纳入34例斑块型银屑病患者，病变部位5 cm外作为健康皮肤对照。研究主要包括以下三个方面：（1）总结银屑病的MPM成像特征，量化银屑病棘层中核周荧光聚集现象。（2）评价各影像特征参数的诊断效能及组合诊断效能。（3）对比MPM与传统反射式共聚焦显微镜（reflectance confocal microscopy，RCM）在银屑病中的诊断效能差别。

结果：与健康皮肤相比，银屑病的MPM特征包括角化不全、颗粒层消失、真皮乳头明亮边缘消失，角质层及表皮厚度增加，细胞核直径增大，这些特征均具有较高的诊断效能（AUC > 0.75，p < 0.001）。此外，94.1%的银屑病患者出现了核周荧光聚集现象（AUC = 0.927，p < 0.001）。平均荧光强度面积值为77 μm²，而健康皮肤荧光强度面积为180 μm²，两者具有显著差异（p < 0.001）。多因素Logistic模型筛选出表皮厚度（OR = 1.245, p = 0.017）、颗粒层存在（OR = 0.013, p = 0.003）及核周荧光聚集（OR =100, p < 0.001）为银屑病的独立预测因子。基于Logistic模型筛选的指标，进行组合诊断效能分析，结果表明表皮厚度 & 颗粒层存在的组合方式诊断效能最高（AUC = 0.956，p < 0.001）。此外，MPM与RCM对银屑病均具有较高的诊断效能（AUC > 0.8），在特征分析中，MPM中的角质层厚度和颗粒层缺失特征有更高的诊断效能。

结论：MPM作为一种无创成像技术，能够进行银屑病的光学活检，提供与病理相似的疾病特征，并能识别独特的核周荧光聚集现象，展现出了优异的诊断效能，显示出了作为银屑病辅助诊断新工具的潜力。

第三部分：皮肤镜及多光子显微镜评估本维莫德治疗银屑病的疗效

背景：银屑病（psoriasis）是一种慢性炎症性皮肤病，对于轻中度银屑病，局部治疗是主要的治疗方式。然而，激素作为传统治疗方式，长期使用会导致表皮萎缩及毛细血管扩张等不良反应。本维莫德（Benvitimod）是一种新型非激素类小分子药物，属于芳香烃受体（aryl hydrocarbon receptor，AhR）调节剂，适用于轻至中度斑块型银屑病，具有良好的安全性。银屑病面积和严重程度指数（psoriasis area and severity index，PASI）为疗效监测的常用评分，但对于轻度面积较小的患者敏感性不足，主观性较强。而多光子显微镜（multiphoton microscopy，MPM）能够在亚微米级别上观察到细胞结构，具有无创成像的优势，适用于临床治疗的监测。

目的：评估皮肤镜及MPM在银屑病治疗过程中对本维莫德疗效的监测能力，探讨MPM其在临床疗效检测中的运用潜力。

方法：本研究为单中心、前瞻性、自身前后对照研究。于2024年2月到2024年7月纳入了24例轻度及中度斑块型银屑病患者。所有患者均接受了为期2月的本维莫德治疗，并在基线（T1）、治疗后4周（T1）、治疗后8周（T2）进行临床评分PASI和目标病灶评分（target lesion score，TLS）以及影像技术监测。探索皮肤镜评分及MPM各影像特征参数治疗前后变化情况。利用Spearman相关分析验证PASI和TLS与MPM可量化特征（表皮厚度、细胞核直径、平均荧光强度面积值）之间的相关性。最后，评价治疗期间的不良反应情况。

结果：经本维莫德8周治疗后，所有临床评分PASI和TLS明显减少60%以上，且皮肤镜评分从6分下降至2分（p < 0.001）。在MPM测量方面，角质层厚度从34 μm下降至26 μm，表皮厚度从116 μm下降至62 μm（p < 0.001）。MPM影像特征方面，经治疗后所有患者角化不全完全消失（0%），颗粒层完全恢复（100%），真皮乳头亮环恢复（80%）（p < 0.001）。通过MPM分析细胞核周荧光的聚集情况，治疗前94.1%的患者出现了荧光聚集，治疗后下降至16.7%，细胞荧光面积从83 μm²增加至233 μm²。表皮厚度，细胞核直径与PASI和TLS呈中等强度的正相关（r= 0.4，p < 0.001），而荧光强度面积与PASI和TLS呈中等强度的负相关（r= -0.4，p < 0.001）。

结论：MPM作为一种无创成像新技术，能够精确量化皮肤结构的变化，有效监测治疗过程中的细胞变化，成为银屑病治疗效果监测的新型工具。

Part Ⅰ: In vivo detection of healthy skin: Comparison of multiphoton

microscopy and reflectance confocal microscopy

Background

In recent years, non-invasive skin imaging technologies have rapidly developed, enabling real-time and in vivo detection of skin diseases. Multiphoton microscopy (MPM), a novel imaging technique with sub-micron resolution, allows cellular-level imaging. Like traditional reflectance confocal microscopy (RCM), MPM also provides cross-sectional imaging of skin layers. However, the clinical application of MPM remains limited, and the advantages of these two devices in skin imaging are still unclear, with a lack of comparative studies between them.

Objective

This study aims to compare the advantages of MPM and RCM in different skin layers, evaluate the differences in skin thickness measurements using both technologies and explore the practical clinical application of MPM, providing a new technological option for clinical skin imaging.

Methods

This single-center, cross-sectional study recruited 56 healthy volunteers from January to March 2023. Three physicians assessed and measured different body areas (cheek, forearm, and back). MPM and RCM were used to evaluate images of various skin layers, including the stratum corneum, stratum granulosum, stratum spinosum, dermo-epidermal junction (DEJ), and superficial dermis. Additionally, differences in epidermal thickness measurements across different body sites were compared between the two instruments. Lastly, the second harmonic generation (SHG) and autofluorescence (AF) signals were utilized to calculate the second harmonic and autofluorescence aging index (SAAID), with multiple linear regression analysis used to examine factors affecting SAAID.

Results

MPM demonstrated superior imaging capabilities for the stratum granulosum, collagen fibers, and elastin fibers, with over 90% of images rated as "excellent" (p < 0.001). RCM, on the other hand, had superior imaging capabilities for the DEJ, with over 90% of images rated as "excellent" (p < 0.001). The epidermal thickness in the cheek was greater than that in the forearm and back. MPM measured a lower average epidermal thickness compared to RCM (RCM: 82 ± 8μm, 76 ± 9μm, 72 ± 9μm for cheek, forearm, and back, respectively; MPM: 72 ± 11μm, 65 ± 9μm, 60 ± 8μm, respectively). Individuals under 40 showed greater epidermal thickness than those over 40 (p < 0.05). There was no significant difference in epidermal thickness between males and females (p > 0.05). The SAAID decreased with age, with a more rapid decline in females, particularly in the cheek area.

Conclusion

MPM, as a novel non-invasive diagnostic technology, shows advantages in imaging the stratum granulosum, elastin fibers, and collagen fibers, while RCM excels in imaging the DEJ. Both devices can accurately measure epidermal thickness, with significant differences observed based on age, gender, and body site. MPM also has an advantage in evaluating skin aging, providing a new clinical evaluation index.

Part Ⅱ: In Vivo Multiphoton Microscopy of Psoriasis: a new disease characteristic analysis technology

Background

Psoriasis is a chronic, systemic inflammatory disease that clinically presents as red plaques covered with silvery scales. The diagnosis and differential diagnosis of psoriasis primarily rely on clinical evaluation and pathological biopsy. Visual assessments are subjective, and skin biopsies may lead to scarring or infection. Therefore, non-invasive optical biopsy holds significant value in the diagnosis and treatment monitoring of psoriasis. Multiphoton microscopy (MPM) is a novel, non-invasive, real-time, in vivo imaging technique that provides sub-micron resolution, enabling observation of single-cell structures and offering layered scanning and three-dimensional imaging from the epidermis to the dermis.

Objective

This study aims to explore the characteristics and diagnostic value of MPM in plaque-type psoriasis, offering a new non-invasive imaging option for psoriasis diagnosing.

Methods

This was a single-center, observational study conducted from February 7 to April 7, 2024, enrolling 34 patients with plaque-type psoriasis. Healthy skin 5 cm away from the lesion was used as a control. The study focused on the following three aspects: (1) summarizing the MPM imaging characteristics of psoriasis and quantifying the perinuclear fluorescence accumulation phenomenon in the psoriatic spinous layer; (2) evaluating the diagnostic efficacy of various imaging feature parameters; (3) comparing the diagnostic efficacy of MPM with traditional reflectance confocal microscopy (RCM) in psoriasis.

Results

Compared to healthy skin, the MPM characteristics of psoriasis included parakeratosis, disappearance of the granular layer, loss of the bright rim, increased thickness of the stratum corneum and epidermis, and enlarged nuclear diameter. All these features demonstrated high diagnostic efficacy (AUC > 0.75, p < 0.001). Additionally, 94.1% of psoriasis patients exhibited the perinuclear fluorescence accumulation phenomenon (AUC = 0.927, p < 0.001), with an average fluorescence intensity area of 77 μm², compared to 180 μm² in healthy skin, showing a significant difference (p < 0.001). A multivariable logistic model identified epidermal thickness (OR = 1.245, p = 0.017), presence of the granular layer (OR = 0.013, p = 0.003), and perinuclear fluorescence accumulation (OR = 100, p = <0.001) as independent predictors of psoriasis. Based on the regression model-selected indicators, the combination diagnostic performance analysis shows that the combination of epidermal thickness & presence of granular layer has the highest diagnostic performance (AUC = 0.956, p < 0.001). Both MPM and RCM demonstrated high diagnostic efficacy (AUC > 0.8), with MPM showing higher diagnostic efficacy for the stratum corneum thickness and granular layer loss features.

Conclusion

MPM, as a non-invasive imaging technique, allows optical biopsy of psoriasis, providing imaging characteristics like pathology and identifying the unique perinuclear fluorescence accumulation phenomenon. It shows excellent diagnostic efficacy. Combined with RCM, it can further enhance the accuracy of psoriasis diagnosis, showing potential as an adjunctive diagnostic tool for psoriasis.

Part Ⅲ: Evaluation of the efficacy of Benvitimod in the treatment of psoriasis using dermoscopy and multiphoton microscopy

Background

Psoriasis is a chronic inflammatory skin disease. For mild to moderate psoriasis, topical treatment remains the primary therapeutic option. However, long-term use of traditional corticosteroids can lead to adverse effects such as epidermal atrophy and capillary dilation. Benvitimod is a new non-steroidal small-molecule drug that acts as an aryl hydrocarbon receptor (AhR) modulator, suitable for the treatment of mild to moderate plaque psoriasis and has shown good safety profiles. The Psoriasis Area and Severity Index (PASI) is commonly used for efficacy monitoring, but it lacks sensitivity for patients with mild and small area lesions and is subject to strong subjectivity. Multiphoton microscopy (MPM), on the other hand, provides sub-micron resolution and allows for cellular structure observation with the advantage of non-invasive imaging, making it suitable for clinical treatment monitoring.

Objective

This study aimed to evaluate the efficacy of skin imaging techniques such as dermoscopy and MPM in monitoring the treatment effects of Benvitimod in psoriasis and explore the potential of MPM for clinical application.

Methods

This study was a single-center, prospective, self-controlled study. Between February and July 2024, 24 patients with mild to moderate plaque-type psoriasis were enrolled. All patients received 2 months of treatment with Benvitimod and were evaluated at baseline (T1), 4 weeks (T2), and 8 weeks (T3) after treatment. Clinical assessments included PASI and Target Lesion Score (TLS), and imaging techniques were used for monitoring. The changes in dermoscopy scores and MPM imaging feature parameters before and after treatment were explored. Spearman correlation analysis was used to verify the relationship between PASI/TLS and quantifiable MPM features (epidermal thickness, nuclear diameter, and average fluorescence intensity area). Finally, adverse events during treatment were assessed.

Results

After treatment with Benvitimod, all clinical scores (PASI/TLS) decreased by more than 50%, and the dermoscopy score decreased from 6 to 2 (p < 0.001). MPM measurements showed a decrease in the stratum corneum thickness from 34 μm to 26 μm and epidermal thickness from 116 μm to 62 μm (p < 0.001). Regarding MPM imaging features, parakeratosis completely disappeared (0%), the granular layer fully recovered (100%), and the dermal papillary bright rim was restored in 80% of patients (p < 0.001). Analysis of the perinuclear fluorescence accumulation with MPM showed that 94.1% of patients exhibited fluorescence aggregation before treatment, which decreased to 16.7% after treatment. The fluorescence area increased from 83 μm² to 233 μm². Epidermal thickness and nuclear diameter showed a moderate positive correlation with PASI/TLS (r = 0.4, p < 0.001), while fluorescence intensity area showed a moderate negative correlation with PASI/TLS (r = -0.4, p < 0.001).

Conclusion

MPM is a noninvasive imaging technique that can accurately quantify changes in skin structure and effectively monitor cellular-level responses during treatment. Thus, it is an important tool for assessing the efficacy of psoriasis treatment.