Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

Design of fluorescent nanocapsules as ratiometric nanothermometers.

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 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; a fluorophore with relatively insensitive fluorescence (rhodamine 640) serves as a reference whereas a sensitive fluorophore (indocyanine green) serves as a sensor. The sensitivity of the nanothermometers depends on the type of nanocapsule-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.

Sensitivity of activatable reactive oxygen species probes by fluorescence spectroelectrochemistry.

We have developed a new analytical method of evaluating activatable fluorescent probes for ROS detection using integrated fluorescence spectroelectrochemistry. The Tafel formalism was applied to describe the process of the probes' oxidation under electrochemical conditions and identify a novel parameter defined as the threshold oxidation potential. This potential can serve as an approximation to the equilibrium potential and can be utilized for determining the sensitivity of a probe to oxidation. Based upon the measured values of threshold potentials, the order of sensitivity towards oxidation among several commonly used probes was determined to be the following (from highest to lowest): 2,7-dihydrodichlorofluorescein > dihydroethidium > dihydrorhodamine 123 > dihydrorhodamine 6G. The presented approach opens up a new direction in synthesizing and screening novel ROS probes with a well-defined sensitivity for in vitro and in vivo applications.

Synthesis of nitric oxide probes with fluorescence lifetime sensitivity.

We present the rationale, synthesis and evaluation of the first activatable fluorescent probe that utilizes fluorescence lifetime change for detection of nitric oxide. The new probe DAP-LT1 features a near-infrared polymethine skeleton with a diaminobenzene functionality incorporated into the meso-position. The probe is partially quenched, and upon reaction with nitric oxide shows an increase in the fluorescence lifetime from 1.08 ns to 1.24 ns.

Minimization of self-quenching fluorescence on dyes conjugated to biomolecules with multiple labeling sites via asymmetrically charged NIR fluorophores.

Self-aggregation of dyes even at low concentrations poses 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.

Multispectral imaging in the extended near-infrared window based on endogenous chromophores.

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.