SWiFi

SWiFi enables wide-field single-molecule fluorescence tracking of fast-diffusing molecules in solution to perform high-throughput, intensity-ratiometric measurements without surface tethering.

Key Features:

• Wide-Field Detection: Allows wide-field imaging of fast-diffusing single fluorophores in solution, extending beyond confocal point-detection and conventional objective-type TIRF geometries.
• Ratiometric Measurements: Supports intensity ratiometric measurements to obtain fluorescence intensity ratios from thousands of individual events per minute.
• High-Throughput Capability: Enables rapid collection of several thousands of individual data points per minute for large-scale single-molecule experiments.
• Versatility Across Samples: Demonstrated with double-stranded DNA (dsDNA) containing FRET pairs, fluorescent nanodiamonds, DNA Holliday junctions, and protein–DNA interactions.

Scientific Applications:

• Bridging Detection Geometries: Bridges confocal and objective-type TIRF detection geometries to expand detection options for single-molecule studies.
• Single-Molecule Dynamics: Enables observation of fast-diffusing molecular mobility and interactions in solution without surface attachment.
• FRET and Conformational Studies: Facilitates high-throughput ratiometric FRET measurements for dsDNA FRET pairs and conformational dynamics such as Holliday junctions.

Methodology:

Operates as an extension to existing objective-type TIRF microscopes, leveraging their optical systems to overcome detection geometry limitations and enable experiments in solution without surface tethering.