Loading...
Optimizing Single-Molecule Blink-Based Multiplexing
Smith, Emma
Smith, Emma
Abstract
Multicolor super-resolved imaging is a powerful technique for visualizing biological structures with unprecedented levels of detail. However, in order to differentiate various fluorophores, they must exhibit distinct emission spectra, thus limiting the set of available probes and adding to experimental demands. To overcome these issues, the Wustholz lab has developed blinking-based multiplexing (BBM), a novel method for differentiating spectrally-overlapped emitters such as rhodamine 6G (R6G) and CdSe/ZnS quantum dots (QD) based solely on their characteristic blinking behavior. Blinking is defined as the fluctuations in emission intensity that occur in individual fluorophores while undergoing continuous photoexcitation via a laser. BBM was previously demonstrated to be effective on a glass substrate at ~1 µW excitation power using emitter concentrations of ~1 nM, but the impact of imaging conditions on classification accuracy are unknown. Here, the impact of excitation power and labeling density on BBM performance for QD and R6G emitters is investigated using a confocal microscope. We demonstrate that excitation power can be tuned to optimize classification accuracy from ~83% at 0.8 µW to ~93% at 1.2 µW. To mimic typical imaging conditions, BBM is evaluated at increased labeling densities. Although classification accuracies of ~93% are maintained, differentiation may be artificially enhanced by aggregation of QD at high concentrations. We then transition to widefield microscopy, using an electron-multiplying charge-coupled device (EMCCD) camera to optimize localization precision for the purposes of creating super-resolved images with BBM.
Description
Date
2024-05-01
Journal Title
Journal ISSN
Volume Title
Publisher
Collections
Download Dataset
Rights Holder
Usage License
Embargo
Research Projects
Organizational Units
Journal Issue
Keywords
Citation
Department
Chemistry