Abstract. To minimize the problem with scattering in deep tissues while increasing the penetration depth, we explored the feasibility of imaging in the relatively unexplored extended near infrared (exNIR) spectral region at 900 to 1400 nm with endogenous chromophores. This region, also known as the second NIR window, is weakly dominated by absorption from water and lipids and is free from other endogenous chromophores with virtually no autofluorescence. To demonstrate the applicability of the exNIR for bioimaging, we analyzed the optical properties of individual components and biological tissues using an InGaAs spectrophotometer and a multispectral InGaAs scanning imager featuring transmission geometry. Based on the differences in spectral properties of tissues, we utilized ratiometric approaches to extract spectral characteristics from the acquired three-dimensional "datacube". The obtained images of an exNIR transmission through a mouse head revealed sufficient details consistent with anatomical structures.
Self-aggregation of dyes even at low concentrations pose a considerable challenge in preparing sufficiently bright molecular probes for in vivo imaging, particularly in the conjugation of near infrared cyanine dyes to polypeptides with multiple labeling sites. Such self-aggregation leads to a significant energy transfer between the dyes resulting in severe quenching and low brightness of the targeted probe. To address this problem, we designed a novel type of dye with an asymmetrical distribution of charge. Asymmetrical distribution prevents the chromophores from π-stacking thus minimizing the energy transfer and fluorescence quenching. The conjugation of the dye to polypeptides showed only a small presence of an H-aggregate band in the absorption spectra and, hence, a relatively high quantum efficiency.
Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(DL-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Čerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.
We have developed a novel design of optical nanothermometers that can measure the surrounding temperature in the range of 20–85°C. The nanothermometers comprise of two organic fluorophores encapsulated in a crosslinked polymethacrylate nanoshell. The role of the nanocapsule shell around the fluorophores is to form a well-defined and stable microenvironment to prevent other factors besides temperature from affecting the dyes’ fluorescence. The two fluorophores feature different temperature-dependent emission profiles where a fluorophore with relatively insensitive fluorescence (rhodamine 640) serves as a reference while a sensitive fluorophore (indocyanine green) serves as a sensor. The sensitivity of the nanothermometers depends on the type of nanocapsules-forming lipid and is affected by the phase transition temperature. Both the fluorescence intensity and the fluorescence lifetime can be utilized to measure the temperature.
Near-infrared heptamethine cyanine dye is functionalized with pyrazole derivatives at the meso-position to induce pH-dependent photophysical properties. The presence of pyrazole unsubstituted at 1-position is essential to induce pH-dependent fluorescence intensity and lifetime changes of these dyes. Replacement of meso-chloro group of cyanine dye IR820 with 1N-unsubstituted pyrazole resulted in the pH-dependent fluorescence lifetime changes from 0.93 ns in neutral media to 1.27 ns in acidic media in DMSO. Time resolved emission spectra (TRES) revealed that at lower pH, the pyrazole consists of fluorophores with two distinct lifetimes, which corresponds to pH sensitive and non-pH sensitive species. In contrast, 1N-substituted pyrazoles do not exhibit pH response, suggesting excited state electron transfer as the mechanism of pH-dependent fluorescence lifetime sensitivity for this class of compounds.
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