Martínez-Zaguilán R, Gurulé M W, Lynch R M
Department of Physiology, University of Arizona, Tucson 85724, USA.
Am J Physiol. 1996 May;270(5 Pt 1):C1438-46. doi: 10.1152/ajpcell.1996.270.5.C1438.
Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of fura 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi and transiently increased [Ca2+]i. Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2-]i. Glucose treatment generally resulted in rapid and sustained increases in both [Ca2+]i and pHi but also heterogeneous pHi and [Ca2+]i responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pHi and [Ca2+]i in single cells.
本文描述了一种微观光谱成像方法，可通过多种离子指示剂的组合光谱同时监测pH值和Ca2+。细胞发出的光聚焦到光栅光谱仪上，光谱由冷却的电荷耦合器件相机成像。分析fura 2和SNARF-1的组合光谱输出，以同时跟踪细胞内Ca2+浓度（[Ca2+]i）和细胞内pH值（pHi）的变化，并校正pHi同时变化时的Ca2+信号。单个仓鼠胰岛素瘤（HIT-T15）细胞对离子稳态效应器的反应是异质性的。用NH4Cl处理可提高pHi并短暂增加[Ca2+]i。去除NH4Cl会导致胞质酸化，同时[Ca2+]i无变化或持续增加。葡萄糖处理通常会导致[Ca2+]i和pHi迅速且持续增加，但pHi和[Ca2+]i反应也存在异质性。对fura 2信号进行pH校正对于跟踪NH4Cl引起的Ca2+转变很重要，但对葡萄糖诱导的反应不太重要。光谱成像显微镜为同时测量单个细胞中的pHi和[Ca2+]i提供了一种灵敏的方法。
