Fluorescence-based imaging has an enormous impact on our understanding of biological systems. However, in vivo fluorescence imaging is greatly influenced by tissue scattering. A better understanding of this dependence can improve the potential of noninvasive in vivo fluorescence imaging. In this article, we present a diffusion model, based on an existing master-slave model, of isotropic point sources imbedded in a scattering slab, representing fluorophores within a tissue. The model was compared with Monte Carlo simulations and measurements of a fluorescent slide measured through tissue-like phantoms with different reduced scattering coefficients (0.5-2.5 mm À1 ) and thicknesses (0.5-5 mm). Results show a good correlation between our suggested theory, simulations and experiments; while the fluorescence intensity decays as the slab's scattering and thickness increase, the decay rate decreases as the reduced scattering coefficient increases in a counterintuitive manner, suggesting fewer fluorescence artifacts from deep within the tissue in highly scattering media.

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, θ 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 θ, together with the steady-state images of fluorescence intensity (FI) and FA (r ). 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 4.01 AE 0.01 to 3.56 AE 0.02 ns, whereas both r and θ were significantly increased from 0.053 AE 0.012 to 0.252 AE 0.003 and 0.15 AE 0.05 to 11.25 AE 1.87 ns, 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 r from 0.100 AE 0.011 to 0.150 AE 0.013 and θ from 0.98 AE 0.13 to 1.65 AE 0.20 ns for the first CDs and from 0.280 AE 0.008 to 0.310 AE 0.004 and 5.55 AE 1.08 to 7.95 AE 0.97 ns 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, r , and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

Although single point time-resolved fluorescence anisotropy (FA) measurements are well established and routinely used for various applications in many laboratories, only a few reports described their extension into two-dimensional (2D) time-resolved FA imaging (TR-FAIM). The ability to perform TR-FAIM can offer cellular imaging based on the rotational correlation time (θ) that depends on the viscosity and dynamic properties of the tissues. We extended existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FAIM, which produces visual maps of θ. The proof of concept of the FD TR-FAIM was validated on 7 fluorescein solutions with increasing viscosities (achieved by increasing glycerol concentration between 0-80%). The studies were performed using images of θ as well as by characterizing the peak (mode) and the full width half maximum (FWHM) of its histograms (of normal probability distribution) and extracting the limiting FA (r0). The θ of the 7 solutions was significantly increased from 0.15±0.05 to 11.25±1.87ns, whereas r0 decreased from 0.40±0.01 to 0.30±0.06. The FD TR-FAIM provides wide-field imaging of the θ of the fluorophore, and hence offers a potential simultaneous interrogation with great sensitivity of diverse chemical and physical phenomena. In addition, as θ can vary according to the local microenvironment and across the sample under investigation, it can characterize different compartments of complex structures such as cells. Through the FD TR-FAIM a large variety of information can be probed from each sample and therefore it may become a reliable and powerful diagnostic tool for cellular imaging and biosensing.