Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media

• Published in: Review of scientific instruments 2024-08, Vol.95 (8)
• Main Authors: Kumar, Amalesh, Bharadwaj, Anupam, Choudhury, Pranjal, Mathew, Sam P., Jaganathan, Bithiah Grace, Boruah, Bosanta R.
• Format: Article
• Language: English
• Subjects: Blinking; Dyes; Excitation; Filaments; Fluorescence; Image acquisition; Image reconstruction; Image resolution; Imaging techniques; Lasers; Signal to noise ratio; Tuning
• ISSN: 0034-6748; 1089-7623; 1089-7623
• 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.