Lee Jun Ho, Lee Seunghun, Yoon Calvin J, Park Jin Hyoung, Tchah Hungwon, Kim Myoung Joon, Kim Ki Hean
Department of Mechanical Engineering, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, South Korea.
Department of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, South Korea.
Biomed Opt Express. 2016 Jan 26;7(2):677-87. doi: 10.1364/BOE.7.000677. eCollection 2016 Feb 1.
Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other.
真菌性角膜炎是由真菌病原体感染角膜所致。基于光学显微镜的诊断方法相比传统微生物学方法，能够实现快速且非侵入性的检查，可能具有一定优势。反射共聚焦显微镜（RCM）和双光子二次谐波产生显微镜（TPSHGM）已应用于真菌性角膜炎的临床前或临床研究。在本报告中，对RCM和TPSHGM在离体真菌性角膜炎兔模型成像中的特性进行了表征和比较。使用两种真菌菌株诱导真菌感染：烟曲霉和白色念珠菌。对感染的角膜在新鲜状态下依次采用这两种方法进行成像，并对图像进行分析。基于形态学，RCM和TPSHGM均可在角膜内检测到这两种真菌菌株：烟曲霉具有独特的丝状结构，白色念珠菌在表面有圆形结构，在角膜基质中有细长结构。这些成像结果得到了组织学的证实。RCM和TPSHGM之间的比较显示出几个特点。虽然RCM和TPSHGM图像之间具有良好的相关性，但由于对比度机制的差异，它们的图像略有不同。由于背景信号较高，RCM对感染浑浊的角膜图像对比度相对较低。与RCM相比，TPSHGM能更清晰地显示角膜中的细胞和胶原蛋白，但需要更高的激光功率来补偿低自发荧光。由于这两种方法提供互补信息，将RCM和TPSHGM结合起来，通过相互弥补各自的弱点，将有助于真菌性角膜炎的检测。
