Thorsrud B A, Harris C
Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109.
Teratology. 1993 Oct;48(4):343-53. doi: 10.1002/tera.1420480408.
A micro-fiberoptic methodology has been developed for non-invasive, real time measurement of endogenous pyridine nucleotide fluorescence from the surface of the visceral yolk sac (VYS) in intact, viable rat conceptuses. Gestational day (GD) 10-12 conceptuses are maintained in a customized perifusion system, which allows for control of oxygenation, as well as the continuous measurement of pH and oxygen concentration in the effluent perifusate. Miniaturized light guides were constructed by drawing 250 microns ESKA acrylic optical fibers through a stainless steel sheath with a high strength epoxy polymer. A single fiber supplied the excitation signal from a mercury arc lamp at a wavelength of 366 nm. The emission signal was returned via three additional fibers, electronically amplified, processed, and recorded, using a dual channel lamp-compensated fluorometer, optimized for detection of reduced pyridine nucleotides at 455 nm. Endogenous fluorescence in the conceptus was monitored by placing the polished tip of the sensor directly on the surface of the VYS. Oxygen-equilibrated conceptuses, exposed to 100% nitrogen, produced a reproducible biphasic surface fluorescence peak, which returned to baseline levels upon reoxygenation of the perifusate. This biphasic response consisted of an initial rapid rise in fluorescence (phase I), followed by an attenuated rate in fluorescence signal increase (phase II). The hypoxia produced age-dependent rates of fluorescence change during phase I, while phase II remained relatively unchanged throughout GD 10-12. These results demonstrate the ability to monitor endogenous fluorescence, non-invasively and in real time, during the period of organogenesis in the intact rat conceptus and will provide valuable information in studies of embryonic metabolism and response to chemical embryotoxicants.
已开发出一种微光纤方法，用于对完整、有活力的大鼠胚胎的内脏卵黄囊（VYS）表面的内源性吡啶核苷酸荧光进行非侵入性实时测量。妊娠第10 - 12天的胚胎被置于定制的灌流系统中，该系统可控制氧合作用，并能持续测量流出灌流液中的pH值和氧浓度。通过将250微米的ESKA丙烯酸光纤穿过带有高强度环氧聚合物的不锈钢护套来制造微型光导纤维。单根光纤提供来自汞弧灯的波长为366 nm的激发信号。发射信号通过另外三根光纤返回，经过电子放大、处理和记录，使用的是双通道灯补偿荧光计，该荧光计针对在455 nm处检测还原型吡啶核苷酸进行了优化。通过将传感器的抛光尖端直接放置在VYS表面来监测胚胎中的内源性荧光。在氧平衡的胚胎中，暴露于100%氮气中会产生可重复的双相表面荧光峰值，当灌流液重新充氧时，该峰值会恢复到基线水平。这种双相反应包括荧光的初始快速上升（阶段I），随后是荧光信号增加速率的衰减（阶段II）。在阶段I中，缺氧产生了与年龄相关的荧光变化速率，而在整个妊娠第10 - 12天期间，阶段II保持相对不变。这些结果表明，能够在完整大鼠胚胎的器官发生期非侵入性地实时监测内源性荧光，这将为胚胎代谢及对化学胚胎毒性物质的反应研究提供有价值的信息。
