Abstract.SignificanceWide-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,θimaging is still rarely investigated in general and in carbon dots (CDs) in particular.AimTo 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 andθ, together with the steady-state images of fluorescence intensity (FI) and FA (r).ApproachThe 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.ResultsThe FLT of fluorescein samples was found to decrease from4.01±0.01to3.56±0.02ns, whereas bothrandθwere significantly increased from0.053±0.012to0.252±0.003and0.15±0.05to11.25±1.87ns, 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 ofrfrom0.100±0.011to0.150±0.013andθfrom0.98±0.13to1.65±0.20nsfor the first CDs and from0.280±0.008to0.310±0.004and5.55±1.08to7.95±0.97nsfor 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.ConclusionsThrough the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT,r, andθ). Nevertheless,θwas the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.