An atomic force microscopy and total internal reflection fluorescence microscopy correlated system (AFM-TIRF) for fluorescence imaging and spectroscopy of a single particle.

Combining atomic force microscopy (AFM) with other optical microscopic techniques is pivotal in nanoscale investigations, particularly leveraging the surface-sensitive properties of total internal reflection fluorescence microscopy (TIRF). A novel design that integrates AFM with a multi-wavelength TIRF is displayed, providing simultaneous fluorescence imaging and spectral acquisition capabilities. We elaborate on the considerations in the instrument design process and demonstrate the performance and potential applications of the instrument through fluorescence imaging and spectroscopy testing of individual nanoparticles. This AFM and TIRF correlated system (AFM-TIRF) emerges as a promising option for single-molecule fluorescence studies, enabling simultaneous manipulation and detection of fluorescence from individual molecules.