Fluorescence lifetime characterization of novel low-pH probes
Fluorescence lifetime characterization of novel low-pH probes
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DOI：
10.1006/abio.2001.5155
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发表时间：
2001-07-15
影响因子：
2.9
通讯作者：
Lakowicz, JR
中科院分区：
文献类型：
--
作者：
Lin, HJ;Herman, P;Lakowicz, JR
细胞酸性区室的结构和功能强烈依赖于跨囊泡膜的pH梯度。囊泡pH的测量和成像需要具有适当pK（a）值的荧光团。在这份报告中，我们的特点是pH值依赖的寿命响应的一个家庭的亲酸探针，LysoSensors，以评估其实用性低pH值的寿命成像。LysoSensors是细胞可渗透的弱碱，其在质子化后选择性地积累在酸性囊泡中。它们在较低的pH范围内具有较高的量子产率，以允许溶酶体的可视化。对于LysoSensors DND-167、DND-189和DND-153，提高缓冲液pH值会增加其碱性侧链的淬灭效应，并显著降低其稳态荧光和寿命。从其寿命响应确定的表观pK值被转移到近中性值，因为其质子化物质的主要强度贡献。LysoSensor DND-189的一个独特特性是其在pH 4和5之间出现的最大值的非单调寿命响应。LysoSensor DND-192在较宽的pH值范围内未显示出显著的寿命变化。LysoSensor DND-160是唯一的激发和发射比率探针，显示出显著的pH依赖性寿命变化及其光谱位移。由于其明亮的碱性形式，从寿命响应确定的其表观pK值与溶酶体pH值相当。由于pH依赖性吸收光谱，表观pKi值可以通过改变激发和/或发射波长在3和5之间操纵。这些结果表明，LysoSensor DND-160是用于确定溶酶体pH的寿命成像的有前途的探针。（C）2001学术出版社。
The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK, values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK, values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK, values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH. (C) 2001 Academic Press.