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Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy
Aleksandra Arsić1, Nevena Stajković1, Rainer Spiegel2
1Werner Reichardt Centre for Integrative Neuroscience, University of Tübingen, Otfried-Müller-Straße 25, 72076, Tübingen, Germany.
Scientific Reports
|April 17, 2020
Summary
Vectashield mounting medium quenches Alexa Fluor 647 (AF647) fluorescence, hindering super-resolution microscopy. A new variant, Alexa Fluor Plus 647, shows promise for overcoming this limitation in direct stochastic optical reconstruction microscopy (dSTORM).
Area of Science:
- Microscopy and Imaging Science
- Biochemistry and Molecular Biology
Background:
- Vectashield is a common mounting medium for microscopy, including super-resolution techniques like direct stochastic optical reconstruction microscopy (dSTORM).
- Alexa Fluor 647 (AF647) is a widely used dye for dSTORM, requiring specific buffer conditions for single-molecule localization and image reconstruction.
- AF647 is known to blink in Vectashield, a prerequisite for dSTORM, but its fluorescence intensity can be quenched in this medium.
Purpose of the Study:
- To quantitatively analyze the fluorescence intensity of AF647 in various imaging media.
- To assess the suitability of Vectashield for dSTORM imaging of both high and low-abundance AF647-labeled targets.
- To evaluate the performance of the new Alexa Fluor Plus 647 dye in dSTORM imaging.
Main Methods:
- Quantitative analysis of AF647 fluorescence intensity across different imaging media.
- Assessment of Vectashield's compatibility with dSTORM for targets with varying labeling densities.
- Comparative performance evaluation of AF647 and Alexa Fluor Plus 647 in dSTORM experiments.
Main Results:
- Vectashield significantly quenches AF647 fluorescence, particularly impacting the visualization of low-abundance targets like neuronal cytoskeleton components.
- This quenching effect compromises image quality in both conventional and dSTORM imaging.
- Alexa Fluor Plus 647 demonstrates improved performance in dSTORM imaging within Vectashield compared to AF647.
Conclusions:
- The fluorescence quenching of AF647 in Vectashield presents a significant challenge for dSTORM imaging, especially for weakly labeled structures.
- The findings highlight the need for careful selection of mounting media and dyes for optimal super-resolution microscopy.
- Alexa Fluor Plus 647 offers a potential solution for improved dSTORM imaging in Vectashield, warranting further investigation.
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