Sud Dhruv, Mehta Geeta, Mehta Khamir, Linderman Jennifer, Takayama Shuichi, Mycek Mary-Ann
J Biomed Opt. 2006 Sep-Oct;11(5):050504. doi: 10.1117/1.2355665.
For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly(dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (> or =600 ps), high spatial resolution (1.4 microm), calibrated detection (0 to 300+/-8 microM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.
首次将一种用于氧敏染料三水合三（2,2'-联吡啶）二氯化钌（RTDP）的荧光寿命校准方法应用于对含有活的C2C12小鼠成肌细胞的聚二甲基硅氧烷（PDMS）生物反应器中的氧水平进行成像。由于其生物相容性和易于操作，PDMS微系统在生物工程应用中被广泛使用。对于这些系统，氧浓度具有重要意义，并且可能在细胞行为和基因表达中发挥重要作用。荧光寿命成像显微镜（FLIM）基于荧光团激发态寿命产生图像对比度，而激发态寿命反映了局部生物化学情况。宽场时域FLIM系统的独特特性包括可调谐激发（337.1至960 nm）、大的时间动态范围（≥600 ps）、高空间分辨率（1.4微米）、校准检测（0至300±8 microM的氧）以及快速的数据采集和处理时间（10秒）。氧水平随着细胞密度的增加而降低，并且与通过模拟生物反应器氧扩散和细胞增殖获得的模型结果一致。在单个生物反应器回路中，FLIM检测到氧水平的空间异质性，变化高达20%。我们所描述的基于荧光寿命的成像方法避免了基于强度的伪影（包括光漂白和浓度变化），并提供了一种在连续细胞培养系统中对氧监测具有高空间分辨率的技术。
