Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

SIGNIFICANCE: Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore’s diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, [Formula: see text] imaging is still rarely investigated in general and in carbon dots (CDs) in particular. AIM: To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and [Formula: see text] , together with the steady-state images of fluorescence intensity (FI) and FA ([Formula: see text]). APPROACH: The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. RESULTS: The FLT of fluorescein samples was found to decrease from [Formula: see text] to [Formula: see text] , whereas both [Formula: see text] and [Formula: see text] were significantly increased from [Formula: see text] to [Formula: see text] and [Formula: see text] to [Formula: see text] , respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of [Formula: see text] from [Formula: see text] to [Formula: see text] and [Formula: see text] from [Formula: see text] to [Formula: see text] for the first CDs and from [Formula: see text] to [Formula: see text] and [Formula: see text] to [Formula: see text] for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. CONCLUSIONS: Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, [Formula: see text] , and [Formula: see text]). Nevertheless, [Formula: see text] was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.