Yang Rui, Gao Qinyi, Lu Xue, Wu Yukun, Zhu Changxin, Han Zhida, Li Chuanya, Wei Mengmeng
School of Electronics and Information Engineering, Changshu Institute of Technology, Changshu 215500, PR China.
School of Electronics and Information Engineering, Changshu Institute of Technology, Changshu 215500, PR China.
Spectrochim Acta A Mol Biomol Spectrosc. 2025 Mar 15;329:125481. doi: 10.1016/j.saa.2024.125481. Epub 2024 Nov 23.
The visualization and subsequent monitoring of apoptosis holds paramount significance in the domains of physiology, pathology, and pharmacology. However, traditional probes require high staining concentrations and multiple washing steps, which would alter the specimen's micro-environment, potentially inducing harm to specimen. To overcome these challenging issues, we have rationally designed and prepared a pH-inert lysosomal probe (named IVTI) to wash-free visualize apoptosis with ultra-low concentration to alleviate the disturbance of probe concentration, washing procedure and pH variations. Compared with general lysosomal probes, IVTI showed a significant fluorescence boost in reflex to elevated viscosity, while its fluorescence intensity remained mostly still when altering pH values, which could achieve more accurate visualization of lysosomes. Moreover, the probe can detect minute viscosity fluctuations in lysosomes under extra-low concentration, greatly eliminating the effect of probe concentration and washing steps to live bio-samples. Furthermore, compared to LTR (Lyso-Tracker Red, a commercial lysosome probe), IVTI offered exceptional imaging capabilities, and the fluorescence images of IVTI was still clear when lysosomal pH increased, which attributed to the pH-inert properties of IVTI. In view of the excellent imaging abilities, the pH-inert probe was applied to in-situ and real time visualize viscosity changes of live cells under extra-low concentration without washing procedure, and the increase of lysosomal viscosity during apoptosis was also monitored by the probe, thereby minimizing the disturbance of probe concentration, washing procedure and pH variations during apoptosis. The probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.
细胞凋亡的可视化及后续监测在生理学、病理学和药理学领域具有至关重要的意义。然而，传统探针需要高染色浓度和多次洗涤步骤，这会改变样本的微环境，可能对样本造成损害。为了克服这些具有挑战性的问题，我们合理设计并制备了一种pH惰性溶酶体探针（命名为IVTI），以超低浓度免洗可视化细胞凋亡，减轻探针浓度、洗涤过程和pH变化的干扰。与一般的溶酶体探针相比，IVTI在粘度升高时荧光显著增强，而在改变pH值时荧光强度大多保持不变，这可以更准确地可视化溶酶体。此外，该探针在超低浓度下能检测到溶酶体中的微小粘度波动，极大地消除了探针浓度和洗涤步骤对活生物样本的影响。此外，与LTR（溶酶体示踪红，一种商业溶酶体探针）相比，IVTI具有出色的成像能力，当溶酶体pH升高时，IVTI的荧光图像仍然清晰，这归因于IVTI的pH惰性特性。鉴于其出色的成像能力，该pH惰性探针被应用于在超低浓度下原位实时可视化活细胞的粘度变化且无需洗涤过程，并且该探针还监测了细胞凋亡过程中溶酶体粘度的增加，从而最大限度地减少了细胞凋亡过程中探针浓度、洗涤过程和pH变化的干扰。该探针在可视化各种生理过程中与溶酶体相关的动态变化方面具有巨大潜力。
