Spectroscopic behavior of bufotenine and bufotenine N-oxide: Solvent and pH effects and interaction with biomembrane models.

Bufotenine is a fluorescent analog of Dimethyltryptamine (DMT) that has been widely studied due to its psychedelic properties and biological activity. However, little is known about its spectroscopic properties in different media. Thus, we present in this work, for the first time, the spectroscopic behavior of bufotenine and bufotenine N-oxide by means of their fluorescence properties. Both molecules exhibit changes in optical absorption and emission spectra with variations in pH of the medium and in different solvents. Assays in the presence of biomembranes models, like micelles and liposomes, were also performed. In surfactants titration experiments, the spectral shift observed in fluorescence shows the interaction of both molecules with pre-micellar structures and with micelles. Steady state anisotropy measurements show that both bufotenine and bufotenine N-oxide, in the studied concentration range, interact with liposomes without causing changes in the fluidity of the lipid bilayer. These results can be useful in studies that aim at searching for new compounds, inspired by bufotenine and bufotenine N-oxide, with relevant pharmacological activities and also in studies that use these molecules as markers of psychiatric disorders.

Spatiotemporal Image Correlation Analysis for 3D Flow Field Mapping in Microfluidic Devices.

Microfluidic devices reproducing 3D networks are particularly valuable for nanomedicine applications such as tissue engineering and active cell sorting. There is however a gap in the possibility to measure how the flow evolves in such 3D structures. We show here that it is possible to map 3D flows in complex microchannel networks by combining wide field illumination to image correlation approaches. For this purpose, we have derived the spatiotemporal image correlation analysis of time stacks of single-plane illumination microscopy images. From the detailed analytical and numerical analysis of the resulting model, we developed a fitting method that allows us to measure, besides the in-plane velocity, the out-of-plane velocity component down to vz ≅ 65 µm/s. We have applied this method successfully to the 3D reconstruction of flows in microchannel networks with planar and 3D ramifications. These different network architectures have been realized by exploiting the great prototyping ability of a 3D printer, whose precision can reach few tens of micrometers, coupled to poly dimethyl-siloxane soft-printing lithography.

Image Cross-Correlation Analysis of Time Varying Flows.

In vivo studies of blood circulation pathologies have great medical relevance and need methods for the characterization of time varying flows at high spatial and time resolution in small animal models. We test here the efficacy of the combination of image correlation techniques and single plane illumination microscopy (SPIM) in characterizing time varying flows in vitro and in vivo. As indicated by numerical simulations and by in vitro experiments on straight capillaries, the complex analytical form of the cross-correlation function for SPIM detection can be simplified, in conditions of interest for hemodynamics, to a superposition of Gaussian components, easily amenable to the analysis of variable flows. The possibility to select a wide field of view with a good spatial resolution along the collection optical axis and to compute the cross-correlation between regions of interest at varying distances on a single time stack of images allows one to single out periodic flow components from spurious peaks on the cross-correlation functions and to infer the duration of each flow component. We apply this cross-correlation analysis to the blood flow in Zebrafish embryos at 4 days after fertilization, measuring the average speed and the duration of the systolic and diastolic phases.

The new fluorescent membrane probe Ahba: a comparative study with the largely used Laurdan.

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.