Versatile high-speed confocal microscopy using a single laser beam.

We present a new flexible high speed laser scanning confocal microscope and its extension by an astigmatism particle tracking velocimetry (APTV) device. Many standard confocal microscopes use either a single laser beam to scan the sample at a relatively low overall frame rate or many laser beams to simultaneously scan the sample and achieve a high overall frame rate. The single-laser-beam confocal microscope often uses a point detector to acquire the image. To achieve high overall frame rates, we use, next to the standard 2D probe scanning unit, a second 2D scan unit projecting the image directly onto a 2D CCD-sensor (re-scan configuration). Using only a single laser beam eliminates crosstalk and leads to an imaging quality that is independent of the frame rate with a lateral resolution of 0.235 µm. The design described here is suitable for a high frame rate, i.e., for frame rates well above the video rate (full frame) up to a line rate of 32 kHz. The dwell time of the laser focus on any spot in the sample (122 ns) is significantly shorter than those in standard confocal microscopes (in the order of milli- or microseconds). This short dwell time reduces phototoxicity and bleaching of fluorescent molecules. The new design opens up further flexibility and facilitates coupling to other optical methods. The setup can easily be extended by an APTV device to measure three dimensional dynamics while being able to show high resolution confocal structures. Thus, one can use the high resolution confocal information synchronized with an APTV dataset.

Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements.

HYPOTHESIS: Understanding the mechanism of intact vesicle deposition on solid surfaces is important for effective utilization of vesicles as active ingredient carriers in applications such as drug delivery and fabric softening. In this study, the deposition of large (davg=12µm) and small (davg=0.27µm) cationic vesicles of ditallowethylester dimethylammonium chloride (DEEDMAC) on smooth and rough anionic cellulose fibers is investigated. EXPERIMENTS: The deposition process is studied quantitatively using streaming potential measurements and spectrophotometric determination of DEEDMAC concentrations. Natural and regenerated cellulose fibers, namely cotton and viscose, having rough and smooth surfaces, respectively, are used as adsorbents. Equilibrium deposition data and profiles of substrate streaming potential variation with deposition are used to gain insights into the fate of vesicles upon deposition and the deposition mechanism. FINDINGS: Intact deposition of DEEDMAC vesicles is ascertained based on streaming potential variation with deposition in the form of characteristic saturating profiles which symbolize particle-like deposition. The same is also confirmed by confocal fluorescence microscopy. Substrate roughness is found to considerably influence the deposition mechanism which, in a novel application of electrokinetic methods, is elucidated via streaming potential measurements.

Microgels at the Water/Oil Interface: In Situ Observation of Structural Aging and Two-Dimensional Magnetic Bead Microrheology.

Stimuli-responsive microgels can be used as stabilizers for emulsions. However, the details of structure and the viscoelastic property of the microgel-laden interface are still not well-known. We synthesized fluorescently labeled microgels and used confocal microscopy to observe their arrangement at the water/oil interface. The microgels aggregated spontaneously at the interface, and the aggregated structure reorganized due to thermal motion. The structure of the interfacial layer formed by microgels depended on the microgel concentration at the interface. We suggest that the structure was controlled by the aggregation and adsorption of microgels at the interface. The interparticle separation between microgels at the interface decreased over time, implying a slow aging process of the microgels at the interface. Magnetic beads were introduced at the interface and used to trigger deformation of the microgel layer. Under compression and shear the microgels in the aggregated structure rearranged, leading to plastic deformation, and some elastic responses were also observed.

Evidence for the extrapulmonary localization of inhaled nitric oxide.

Inhaled nitric oxide (NO) has emerged as a promising pulmonary vasodilator to treat pulmonary hypertension associated with heart disease and ventilation/perfusion mismatching. However, the pharmacokinetics of inhaled NO still remains obscure and its cardiopulmonary selectivity appears to be increasingly under debate. In the present study measured NO content and levels of cyclic guanosine 3',5'monophosphate (cGMP), a mediator of NO-induced vasodilation, in a variety of organs from rats subjected to NO inhalation. Electron spin resonance spectroscopy associated to a spin trapping technique using N-methyl D-glucamine dithiocarbamate (FeMGD) was used to directly quantify NO levels in the lung, kidney, liver, aorta, and heart from anesthetized Wistar rats subjected to various doses (0, 20, 50, 100, or 200 ppm) and various times (0, 30, 45, or 75 minutes) of inhaled NO. Inhaled NO at a dose of 100 and 200 ppm significantly increased the NO-FeMGD complex in all organs studied. An increase of cGMP was detected in the lung and the aorta after inhaled NO for 45 minutes at the dose of 50 ppm. No changes in NO levels and its metabolites were shown between 30 and 75 minutes of inhaled NO. The results show that inhaled NO at a dose of 100 ppm or more increases NO levels in other organs beside the lung, strongly suggesting that inhaled NO would be more than a pulmonary vasodilator and its selectivity remains to be reconsidered when used for therapeutic purposes.

Glucose insulin potassium infusion improves systolic function in patients with chronic ischemic cardiomyopathy.

OBJECTIVE: We assessed the effects of glucose-insulin-potassium (GIK) by echocardiography in stable patients with ischemic dysfunction. METHODS: Twelve male patients with stable coronary disease (SCD) and ejection fraction (EF) <45% were studied for systolic function. GIK (glucose 30%, 300 insulin units and KCl 6 g/l) was infused at 1 ml/kg per h over 20 min. Hemodynamic and echocardiographic measurements were recorded at rest (T(0)), at the end (20 min) of GIK infusion (T+20), 20 and 40 min after the end of the infusion (T+40 and T+60). RESULTS: At T+20, a significant decrease in WMSI (wall motion score index) was observed compared with T(0) (2.16+/-0.14 vs. 2.30+/-0.16: P<0.05). An increase in EF was reported at T+40 and T+60 compared with T(0) (44.1+/-7.8% and 53.3+/-11.6% vs. 35.6+/-4.5%, respectively: P<0.01). A decrease in WMSI was observed at T+40 and T+60 compared with rest (2.02+/-0.17 and 1.93+/-0.11 vs. 2.30+/-0.16, respectively: P<0.01). CONCLUSION: Our present work suggests that GIK infusion improves systolic function in patients with SCD and ejection fraction <45%. Further studies are needed to determine if short-term GIK infusion could be useful for therapeutic or diagnostic strategies in these patients.