Piston D W, Masters B R, Webb W W
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
J Microsc. 1995 Apr;178(Pt 1):20-7. doi: 10.1111/j.1365-2818.1995.tb03576.x.
Three-dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide-adenine dinucleotide, NADH, and reduced nicotinamide-adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 microns thickness of the cornea. Two-photon excitation scanning laser microscopy with near-infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 microns within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two-photon microscopy for future redox fluorescence imaging of cellular metabolic states.
通过对天然存在的还原型吡啶核苷酸（还原型烟酰胺腺嘌呤二核苷酸，NADH，以及还原型烟酰胺腺嘌呤二核苷酸磷酸，NADPH，此处统称为NAD(P)H）的荧光进行成像，获得了兔角膜原位细胞代谢氧化/还原状态的三维图谱。在整个400微米厚的角膜中，都获得了横向分辨率达亚微米级的自发荧光图像。采用近红外激发的双光子激发扫描激光显微镜具有高荧光收集效率、减少光损伤以及消除紫外色差等优点，而这些问题以前都会降低吡啶核苷酸荧光的可视化效果。角膜基底上皮（角膜内40微米处）清晰的自发荧光图像显示出大量亚细胞细节，具备监测自发荧光强度随时间变化的能力，这种变化反映了维持眼表所需的氧化代谢和细胞动态变化。通过氰化物暴露证实自发荧光大多源自NAD(P)H，氰化物使角膜中所有细胞类型的荧光增强了约两倍。仅在氰化物处理后才观察到基质中单个角膜细胞的自发荧光图像，而在主要的细胞外胶原蛋白（占基质体积的90%以上）中，荧光与背景无法区分。对角膜细胞代谢的观察证明了双光子显微镜在未来细胞代谢状态氧化还原荧光成像方面的敏感性。
