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

Abstract

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.

Probing Polarity and pH Sensitivity of Carbon Dots in Escherichia coli through Time-Resolved Fluorescence Analyses

“…The derivation of compensation formulas and determination of the G factor (1.05 ± 0.01) are described in Appendix A . The determination of the 5% leakage was previously described elsewhere [ 39 ]. Additionally, excitation with unpolarized light resulted in no phase shift difference between the two polarized components, eliminating the need for corrections for instrumental effects on the phase shift.…”

“…The coefficients 1.02 and 10/9 are derived from the transmission efficiency of the PBS for the transmitted component and for the reflected component (PBS and mirror) . The AC leakage can be compensated using the following equations: and the phase leakage where The full derivation of the DC and AC compensation formulas, as well as the determination of the 5% leakage was previously described elsewhere [ 39 ]. By applying this correction, the impact of the leakage can be mitigated, allowing for an accurate determination of the DC and AC intensity ratios and obtaining reliable maps of r and .…”

“…The terms m m s and m r s are similarly described for the sample. For the FD TR-FAIM calibration, in order to correct for the 5% leakage in the reflected beam PBS (I ⊥ ) in our system from the other polarization component I || , the following equation can be used: The full derivation of the DC and AC compensation formulas, as well as the determination of the 5% leakage was previously described elsewhere [39]. By applying this correction, the impact of the leakage can be mitigated, allowing for an accurate determination of the DC and AC intensity ratios and obtaining reliable maps of r and θ.…”

“…Recently, we reported a FD TR-FAIM system to extract the maps of several fluorescence characteristics: FI, FLT, FA, and θ of two types of CDs. The link of CDs to gold nanoparticles was detected by increased FA and θ [ 39 ]. In this study, we examined the pH- and polarity-responsive properties of newly developed nanomaterials like CDs through FLIM analysis.…”