背景:双分子荧光互补(BiFC)测定通常用于研究蛋白质-蛋白质相互作用。虽然已经开发了几种BiFC检测系统,有一个有限的研究集中在使用激光扫描共聚焦显微镜(LSCM)技术观察原生质体。与原始细胞状态相比,原生质体更容易受到损伤和不稳定的影响,这归因于它们所经历的准备处理,这使得研究人员在LSCM下观察期间操纵它们变得具有挑战性。因此,在BiFC分析中正确有效地利用显微镜技术至关重要。
结果:当目标荧光较弱时,原生质体中叶绿体颗粒的自发荧光会干扰位于核区的BiFC信号的检测。光谱分析表明,叶绿体自发荧光可以被各种类型的激光激发,在大约660nm处观察到最高的荧光信号。此外,我们对不同移液管吸头对原生质体样品完整性的影响的调查表明,利用具有较大开口的切割吸头可以减轻细胞破损。我们介绍了用于研究原生质体BiFC的LSCM技术的工作流程,并讨论了样品制备和图像捕获中涉及的显微镜操作。
结论:当BiFC信号微弱时,它们可能受到叶绿体自发荧光的影响。然而,如果使用得当,叶绿体的自发荧光可以作为有效区分其他信号的极好的内部标记。结合其他发现,本研究可为研究者进行BiFC检测及相关研究提供有价值的参考。
BACKGROUND: The bimolecular fluorescence complementation (
BiFC) assay is commonly used for investigating protein-protein interactions. While several
BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in
BiFC assays.
RESULTS: When the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of
BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast
BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing.
CONCLUSIONS: When the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.