Over the years, an increase in mass plastic production has caused growing concerns about the adverse effects that plastic nanoparticles (NPs) in the environment may have on human health. There are many knowledge gaps regarding the effects that NPs have on human health. Accurate studies prove challenging due to the hurdles in obtaining reliable model particles, performing accurate in vitro toxicology assessments, and visualizing results accurately. Numerous studies published in peer-reviewed literature have used commercially available NPs to represent environmental nanoplastic pollution. The commercial NPs with fluorescent tags were often used since they are easy to be monitored in cellular and organismal studies. However, the legitimacy of these commercial NP products has been questioned due to concerns about surface modifications altering interactions at the nano-bio interfaces, and the possibility that surface-bound fluorophores may detach and result in optical artifacts. Additionally, polystyrene is not the only polymer that should be investigated. We synthesized an orange, fluorescent organic dye and incorporated the dye into PMMA NPs to investigate skin cell uptake and in vivo biodistribution in a fish embryo model. We also compare the uptake results with that of the commonly used polystyrene particles and discuss possible mechanisms of uptake. Results revealed that 1) PMMA NPs can pass into embryos and potentially accumulate in larval bodies, and 2) commercially available sulfate-modified NPs and PMMA NPs accumulate similarly in fluorescently labeled fibroblast cells, however, PMMA NPs accumulate more localized intracellularly.

Point-of-care (POC) measured glomerular filtration rate (GFR) has been a goal of nephrologists for the last 30 years. To this end, a fluorescent GFR tracer agent, relmapirazin, with appropriate photophysical properties for transdermal detection has been rationally designed. Twenty-four standard nonclinical assays to evaluate toxicity as required by the FDA have been performed. Fluorescent detection instrumentation to acquire and process the emission signal from the agent through the skin has been developed. The pilot clinical study objectives were to: • Demonstrate relmapirazin is a GFR agent in humans by comparison to a known non-fluorescent standard agent, • Establish that the GFR as measured by the transdermal fluorescence excretion rate matches the standard labor-intensive non-POC plasma GFR. This combination product was evaluated on 120 subjects covering three clinical sites. Subjects were enrolled spanning normal to very impaired renal function, and for all six skin colors on the Fitzpatrick Skin Scale. Relmapirazin and the standard agent were intravenously administered in consecutive boluses. Prior to dosing, the transdermal sensor was placed on the chest of each subject and fluorescent readings were initiated. The plasma-derived GFR measured from relmapirazin matched the plasma-derived GFR measured from the standard agent. An algorithm was developed to convert the transdermal fluorescence measurement directly into a GFR, applicable to the entire GFR range and for all skin colors. No serious nor significant adverse events were reported. Clinically amenable point-of-care measured GFR has been demonstrated for subjects with a range of GFR values and for all skin colors.

Multicellular aggregates constitute important 3-dimenstional (3D) models for investigating cellular behaviors, including proliferation and migration, that are relevant to neoplastic growth and progression. Studies point to the role of the aggregates’ biomechanical environment in modulating cell proliferation, segregation, and migration. To obtain measurements of mechanical responses at the cellular level, we investigate the use of molecular tension probes in multicellular aggregates. While these tension probes have been used in 2-dimensional monolayers of cells, the feasibility of their application in 3D multicellular aggregates has not been demonstrated yet. In this paper, we utilize a previously described, frequency-domain fluorescence lifetime microscope (FLIM), to test the feasibility of measuring FRET using the Vinculin Tension Sensor (Vinculin TS), in multicellular aggregates of CHO-K1 cells. Our data suggest that the probes can be expressed in the spheroids and that we can measure FLIM-FRET signal from calibration constructs that are diffusely distributed within the cells. When the cells express Vinculin TS, we were able to discern changes in fluorescence distribution, compared with the calibration constructs, with evidence of punctate staining suggestive of localization at adhesion sites. The fluorescence lifetime of puncta expressing Vinculin TS was 2.46 +/- 0.12 ns, longer than 2.21 +/- 0.103 ns, the lifetime of puncta expressing Tail-less Vinculin TS (VinTL), which lacks the actin-binding domain. The longer lifetime is consistent with higher tension across vinculin TS compared with the unloaded VinTL control. Work is underway to fully characterize these puncta.

Fluorescence lifetime-based technology has experienced a rapid growth during the last decade, becoming a unique tool for performing real-time, non-invasive analysis in a large variety of scientific fields, from life-sciences to medical surgery and agrifood. Its potential has aroused the interest of industry and a technological shift in the detection and estimation of fluorescence lifetime is around the corner. Nevertheless, devices capable of implementing state-of-the-art fluorescence lifetime analysis that are compact, customizable and affordable, are still incredibly hard to find on the market. We aim at supporting this existing technological shift by developing instruments that are portable and extremely easy to use, either for beginners or experienced users. Our mission is rooted in the intention of democratizing the use of fluorescence lifetime in both scientific and industrial environments, by creating cutting-edge, AI-driven instruments, with a focus on simplicity and modularity. The idea is to be part of a new, emerging scientific force that has as its leading concept the widespread use of fluorescence lifetime analysis, either as a standing-alone technique or combined with pre-existing setups and instruments. Our instruments will provide a larger number of researchers with access to a technology that is reliable and simple to implement.

Absorption and fluorescence spectra are central to the photosciences. The PhotochemCAD initiative, aimed at assembling digital databases of absorption and fluorescence spectra for use across the photosciences, now comprises <1000 absorption spectra and <500 fluorescence spectra for ~1000 compounds along with companion photophysical parameters (molar absorption coefficient, fluorescence quantum yield) and citations to the originating scientific literature. The spectral databases have chiefly been assembled by digitizing spectra from the vast print literature. The conversion of print spectra to digital form presents several technical challenges: (1) print spectra are plagued by line crossing from overlaid spectra, use of discontinuous lines, and interference from grids, tick marks, and the graph baseline; (2) the print spectrum in some cases lacks wavelength markers or is annotated with markers at odds with values in the accompanying text; and (3) the digitized spectra often are composed of data with uneven wavelength (x-axis) intervals. Here, manually assisted digitization – wherein the user steps through a print spectrum with hand-eye assistance to create the corresponding digital dataset – is compared with automated digitization. Graphical features that bear on use of each method are outlined. Two spreadsheet-based tools have been developed for application following digitization: (i) conversion of xy-coordinate data from uneven to uniform x-axis intervals, and (ii) calibration of the digitized spectrum with appropriate wavelength values. The two tools enable more accurate rendition of print spectra into digital form, as required for qualitative comparisons and quantitative calculations, and have been added to the PhotochemCAD website (http://www.photochemcad.com).