Martin G R, Jain R K
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Microvasc Res. 1993 Sep;46(2):216-30. doi: 10.1006/mvre.1993.1048.
The fluorescence ratio imaging technique is currently applied in vitro and in vivo to measure intracellular as well as extracellular pH. To date, however, no effort has been made to assess the technique's ability to detect spatial pH gradients in vitro or in vivo, where pH gradients are known to exist, in a controlled environment using an independent pH measurement system. We present here both in vitro calibration and in vivo application of fluorescence ratio imaging to measure pH gradients in a biologically useful pH range (6.2-7.8). Isoelectric focusing in short polyacrylamide gels was used to establish spatial pH gradients ranging from approximately 0.2 to 0.5 pH units/mm. The pH gradients were measured independently with fluorescence ratio imaging microscopy (FRIM) and a surface electrode. The average difference between measurements using the two techniques was 0.043 pH units for 10-mm polyacrylamide gels (100 measurements in 12 gels) and 0.046 pH units for 20-mm gels (114 measurements in 15 gels). These gel studies have demonstrated the use of isoelectric focusing to calibrate FRIM for measuring spatial pH gradients characteristic of those found in tumors in vivo. The feasibility of FRIM was tested in vivo by measuring pH in a solid tumor (VX2 carcinoma) and surrounding normal granulation tissue grown in the rabbit ear chamber. The average (+/- SD) pH of normal and neoplastic regions of the composite tissue was 7.18 +/- 0.11 (n = 158) and 6.75 +/- 0.10 (n = 191), respectively. The pH varied by an average of approximately 0.6 pH units over the 5-mm diameter of the chamber and showed a sharp difference at the interface of the two tissue types. These pH values are consistent with the current pH data on normal and neoplastic tissues. Thus FRIM should permit noninvasive in vivo evaluation of new pH-modifying agents and, in combination with other optical techniques, should offer novel mechanistic information about tumor pathophysiology.
荧光比率成像技术目前已应用于体外和体内，用于测量细胞内以及细胞外的pH值。然而，迄今为止，尚未有人努力评估该技术在体外或体内检测空间pH梯度的能力，已知在这些环境中存在pH梯度，且使用独立的pH测量系统在可控环境下进行检测。我们在此展示了荧光比率成像技术在体外校准和体内应用，以测量生物有用pH范围（6.2 - 7.8）内的pH梯度。使用短聚丙烯酰胺凝胶中的等电聚焦来建立范围约为0.2至0.5个pH单位/毫米的空间pH梯度。使用荧光比率成像显微镜（FRIM）和表面电极独立测量pH梯度。对于10毫米聚丙烯酰胺凝胶，两种技术测量结果的平均差异为0.043个pH单位（12块凝胶中的100次测量），对于20毫米凝胶，平均差异为0.046个pH单位（15块凝胶中的114次测量）。这些凝胶研究证明了使用等电聚焦来校准FRIM，以测量体内肿瘤中存在的特征性空间pH梯度。通过测量兔耳腔中生长的实体瘤（VX2癌）和周围正常肉芽组织的pH值，在体内测试了FRIM的可行性。复合组织正常和肿瘤区域的平均（±标准差）pH值分别为7.18±0.11（n = 158）和6.75±0.10（n = 191）。在腔室直径5毫米范围内，pH平均变化约0.6个pH单位，并且在两种组织类型的界面处显示出明显差异。这些pH值与当前关于正常和肿瘤组织的pH数据一致。因此，FRIM应该能够对新的pH调节药物进行非侵入性体内评估，并且与其他光学技术相结合，应该能够提供有关肿瘤病理生理学的新机制信息。
