Kumar Amalesh, Bharadwaj Anupam, Choudhury Pranjal, Mathew Sam P, Jaganathan Bithiah Grace, Boruah Bosanta R
Department of Physics, IIT Guwahati, Guwahati 781039, Assam, India.
Department of Bioscience and Bioengineering, IIT Guwahati, Guwahati 781039, Assam, India.
Rev Sci Instrum. 2024 Aug 1;95(8). doi: 10.1063/5.0217409.
Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638 nm. An excitation power density of 0.60 kW/cm2 at 638 nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.
超分辨率成像技术通过突破衍射极限并实现亚细胞细节的可视化，从根本上改变了我们对细胞结构和动力学的理解。流行的超分辨率方法——随机光学重建显微镜（STORM）依赖于单个荧光分子的精确定位。最近人们探索了将Vector Shield用作直接STORM超分辨率成像方案的封片剂的重要性。最常用的染料之一Alexa Fluor 647（AF647）在Vector Shield中表现出明显的闪烁现象。然而，为了观察荧光团的显著闪烁以重建超分辨图像，需要调整激发激光的功率。这项工作展示了在样品平面中调整激发功率密度，以使用Vector Shield中的AF647实现卓越的成像性能。实验使用了包含MDA-MB-231乳腺癌细胞系的样品。细胞的肌动蛋白丝用与鬼笔环肽偶联的AF647染料染色。在实验中，我们使用了一个低成本的基于openFrame的STORM系统，该系统配备了一个由Arduino可编程调节的638 nm激光源。观察到在样品平面中638 nm处的激发功率密度为0.60 kW/cm²时，可在图像采集期间最大化信噪比、切换事件数量和每个事件检测到的光子数量，从而在分辨率方面实现最佳成像性能。这项工作的成果将推动在细胞生物学中进一步应用基于STORM的超分辨成像，使用Vector Shield中的Alexa Fluor 647。
