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, 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±0.01 to 3.56±0.02 ns, whereas both r and θ were significantly increased from 0.053±0.012 to 0.252±0.003 and 0.15±0.05 to 11.25±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±0.011 to 0.150±0.013 and θ from 0.98±0.13 to 1.65±0.20 ns for the first CDs and from 0.280±0.008 to 0.310±0.004 and 5.55±1.08 to 7.95±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.

Fluorescence attenuated by a thick scattering medium: Theory, simulations and experiments.

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-1 ) 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.

Classification of fluorescent anisotropy decay based on the distance approach in the frequency domain.

Frequency-domain (FD) fluorometry is a widely utilized tool to probe unique features of complex biological structures, which may serve medical diagnostic purposes. The conventional data analysis approaches used today to extract the fluorescence intensity or fluorescence anisotropy (FA) decay data suffer from several drawbacks and are inherently limited by the characteristics and complexity of the decay models. This paper presents the squared distance (D2) technique, which categorized samples based on the direct frequency response data (FRD) of the FA decay. As such, it improves the classification ability of the FD measurements of the FA decay as it avoids any distortion that results from the challenged translation into time domain data. This paper discusses the potential use of the D2 approach to classify biological systems. Mathematical formulation of D2 technique adjusted to the FRD of the FA decay is described. In addition, it validates the D2 approach using 2 simulated data sets of 6 groups with similar widely and closely spaced FA decay data as well as in experimental data of 4 samples of a fluorophore-solvent (fluorescein-glycerol) system. In the simulations, the classification accuracy was above 95% for all 6 groups. In the experimental data, the classification accuracy was 100%. The D2 approach can help classify samples whose FA decay data are difficult to extract making FA in the FD a realistic diagnostic tool. The D2 approach offers an advanced method for sorting biological samples with differences beyond the practical temporal resolution limit in a reliable and efficient manner based on the FRD of their time-resolved fluorescence measurements thereby achieving better diagnostic quality in a shorter time.