Lingotti Gabriel, Jones Mark R
School of Science, Western Sydney University, Penrith, New South Wales, Australia.
Biochem Biophys Rep. 2025 Feb 20;41:101956. doi: 10.1016/j.bbrep.2025.101956. eCollection 2025 Mar.
Ratiometric and single-wavelength fluorophores are limited in their ability to provide basal level activity prior to treatment. This study presents a novel approach to characterise cell-wide basal activity of calcium using first and second harmonic spectral phasor analysis. Cells stained with the single-wavelength calcium fluorophore Oregon Green™ BAPTA 1-AM exhibited significant differences in wavelength or width in the nucleus, cytoplasm and membrane under basal conditions. Large and small cursor analysis was applied in the first and second harmonic, with smaller cursors revealing a region of interest enveloping and protruding from the nucleus in a structure akin to the sarcoplasmic reticulum. The first harmonic was found to be more sensitive in measurements of , while the second harmonic showed increased sensitivity in measurements of spectral width. The results of this study indicate that first and second harmonic frequencies should be used in conjunction with phasor analysis of fluorophore microenvironments, rather than the first harmonic alone. Use of this approach may provide more insight into the cellular microenvironment under basal activity and treatment responses.
比率型和单波长荧光团在治疗前提供基础水平活性的能力有限。本研究提出了一种利用基波和二次谐波光谱相量分析来表征细胞整体钙基础活性的新方法。用单波长钙荧光团俄勒冈绿™ BAPTA 1-AM染色的细胞在基础条件下，细胞核、细胞质和细胞膜中的波长或宽度存在显著差异。在基波和二次谐波中应用了大小光标分析，较小的光标揭示了一个类似于肌浆网的结构中包围细胞核并从细胞核突出的感兴趣区域。发现基波在测量[此处原文缺失具体测量内容]时更敏感，而二次谐波在光谱宽度测量中显示出更高的灵敏度。本研究结果表明，基波和二次谐波频率应与荧光团微环境的相量分析结合使用，而不是仅使用基波。使用这种方法可能会为基础活性和治疗反应下的细胞微环境提供更多见解。
