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, θ imaging 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 and θ, 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 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.ConclusionsThrough 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.
The unique fluorescent nanomaterials known as carbon dots (CDs) are highly resistant to photobleaching, have low toxicity, and are well soluble in water. Polyethyleneimine (PEI) coated CDs are a novel fluorophore with good biocompatibility and pH sensing ability. Here, p-phenylenediamine (p-PD) is used as a carbon source and hyperbranched PEI is used as a surface passivation agent in a simple, one-step hydrothermal synthesis process. The CDs optical characteristics are pH-responsive due to the presence of different amine groups on PEI, which is functional polycationic polymer. The limits of techniques based on fluorescence intensity can be overcome by fluorescent lifetime imaging microscopy (FLIM), a very sensitive method for detecting a microenvironment. In this study, FLIM was used to measure pH with pH-sensitive CDs. These molecules are nontoxic to the cells, and the positively charged CDs have the potential for nuclear targeting, allowing for electrostatic contact with DNA in the nucleus. Higher wavelengths have a larger penetration depth of electromagnetic radiation and low tissue autofluorescence, hence CDs emitting at these wavelengths are used for biolabeling applications. However, the quantum yield of these synthesized red-emissive CDs is lower. In order to enhance it, they are conjugated with gold nanoparticles(AuNPs) for metal enhanced fluorescence (MEF). Through a potent covalent bond between them, the AuNPs are linked to CDs surfaces. These gold-CDs nanoconjugate can be used in the future for targeted imaging applications.
In today’s research area it is extremely important to assemble nanomaterials into electric devices at the nanoscale level due to the rapid expansion of nanotechnology in various fields. Designing a nanohybrid composed of gold nanoparticles (AuNPs) and red-emitting carbon dots (CDs) can be used to develop a fluorescence lifetime imaging (FLIM) based logic gate that can respond to multiple input parameters. The AuNPs are conjugated to CDs surfaces through a strong covalent linkage between them. These fluorescence lifetimes-based logic gates could be the new way to overcome the limitation of fluorescence intensity-based logic gates. The Au-CDs nanohybrid shows significant fluorescence quenching of pristine CDs after conjugation of gold nanoparticles. This quenched fluorescence can be recovered back by using a proper recovering agent giving us a reversible logic output. This nanohybrid can be used to construct complex logic functions as the fluorescence logic output is independent of concentration and excitation source.
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