TiO2 Nanofibrous Interface Development For Raman Detection of Environmental Pollutants

Sensor development has been reliant on planar Au and Ag nanoparticle research. The current findings explored a unique 3-D network of crystalline TiO2 nanoparticles linked as nanofibers. In addition to the favorability of using TiO2 for chemical and bio-molecular sensing, the nanofiber network provides molecular diffusion control and an increased confocal volume signal. Controlled femtosecond laser synthesis is also demonstrated that directly impacts surface-enhanced Raman spectroscopy detection of two common environmentally harmful chemicals: bisphenol A and diclofenac sodium salt. These findings assert that 3-D nanofibrous network porosity optimization is crucial for Raman monitoring of drinking water.

TiO2 Nanofibrous Interface Development For Raman Detection of Environmental Pollutants

Sensor development has been reliant on planar Au and Ag nanoparticle research. The current findings explored a unique 3-D network of crystalline TiO2 nanoparticles linked as nanofibers. In addition to the favorability of using TiO2 for chemical and bio-molecular sensing, the nanofiber network provides molecular diffusion control and an increased confocal volume signal. Controlled femtosecond laser synthesis is also demonstrated that directly impacts surface-enhanced Raman spectroscopy detection of two common environmentally harmful chemicals: bisphenol A and diclofenac sodium salt. These findings assert that 3-D nanofibrous network porosity optimization is crucial for Raman monitoring of drinking water.

CALIBRATION OF A TABLETOP CONFOCAL MICROBEAM X-RAY FLUORESCENCE SPECTROMETER FOR A QUANTITATIVE DEPTH PROFILES EVALUATION

Confocal micro-beam X-ray fluorescence analysis (confocal micro-XRF) is a non-destructive analytical tool for investigation of sample composition that enables acquiring three-dimensionally resolved information. This work describes a calibration procedure of a laboratory confocal micro-XRF setup, which leads to determination of its characteristic parameters. The calibration is performed using a tabletop confocal micro-XRF spectrometer designed recently at the Czech Technical University in Prague. The calibration procedure performed within this work comprises the essential steps of the setup characterization: excitation spectrum calculation, experimental determination of energy-dependent confocal volume size and integral sensitivity and calculation of the spectrometer sensitivity function. The results of the setup calibration will be used for development of a procedure enabling quantitative evaluation of the measured depth profiles.

Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means–such as fluorescence correlation spectroscopy (FCS) and related techniques–they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

Human telomere repeat binding factor TRF1 replaces TRF2 bound to shelterin core hub TIN2 when TPP1 is absent

Human telomeric repeat binding factors TRF1, TRF2 along with TIN2 form a core of shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence Cross-Correlation Spectroscopy (FCCS) was used to describe the complex formation by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substituted TRF2 on TIN2. We assessed the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces allosteric changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.

A fluorescence correlation spectrometer for measurements in cuvettes

ABSTRACTWe have developed a fluorescence correlation spectroscopy (FCS) setup for performing single molecule measurements on samples inside regular cuvettes. We built this by using an Extra Long Working Distance (ELWD), 0.7 NA, air objective with working distance > 1.8 mm. We have achieved counts per molecule > 44 kHz, diffusion time < 64 μs for rhodamine B in aqueous buffer and a confocal volume < 2 fl. The cuvette-FCS can be used for measurements over a wide range of temperature that is beyond the range permitted in the microscope-based FCS. Finally, we demonstrate that cuvette-FCS can be coupled to automatic titrators to study urea dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette-FCS in the experiments that are regularly performed in fluorimeters but are generally avoided in microscope-based FCS.

A simulation-guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase

Fluorescence correlation spectroscopy (FCS) is a single molecule based technique to temporally resolve rate-dependent processes by correlating the fluorescence fluctuations of individual molecules traversing through a confocal volume.