Optical differential temperature measurement with beat frequency phase fluorometry.

We present, to the best of our knowledge, a new method for differential temperature measurement based on thermal sensitivity of the fluorescence lifetime of thermographic phosphors. Pairs of thermographic phosphors are excited with intensity-modulated light at frequencies ω and ω+Δω. The phase shift Δθ of the summary fluorescence intensity beat signal envelope is measured.

Interference filter tilting to detect a polycyclic aromatic hydrocarbon at the second harmonic of wavelength modulation frequency.

We present a practical implementation of the wavelength-modulation spectroscopy technique with second-harmonic detection for selective detection and concentration measurements of polycyclic aromatic hydrocarbons (PAHs)-common fossil fuel pollutants. The method is based on excitation light wavelength modulation around the maximum of a narrow peak in the absorption spectrum of a PAH and lock-in detection of fluorescence at the second harmonic of the excitation wavelength modulation frequency. A violet LED is used as the excitation source and wavelength modulation is performed via narrowband interference filter tilting.

Spectral shift mechanisms of chlorophylls in liquids and proteins.

Origins of non-excitonic spectral shifts of chlorophylls that can reach -1,000 cm(-1) in pigment-protein complexes are actively debated in literature. We investigate possible shift mechanisms, basing on absorption and fluorescence measurements in large number of liquids. Transition wavelength in solvent-free state was estimated (±2 nm) for chlorophyll a (Chl a, 647 nm), Chl b (624 nm), bacteriochlorophyll a (BChl a, 752 nm), and pheophytines.

Gaussian beam reflection and refraction by a spherical or parabolic surface: comparison of vectorial-law calculation with lens approximation.

A ray-tracing approach is used to demonstrate efficient application of the vectorial laws of reflection and refraction to computational optics problems. Both the full width at half-maximum (fwhm) and offset of Gaussian beams resulting from off-center reflection and refraction are calculated for spherical and paraboloidal surfaces of revolution. It is found that the magnification and displacement depend nonlinearly on the miscentering.

Cataract diagnosis by measurement of backscattered light.

We present a portable optical cataract assessment technology which measures with a circular photodetector the fraction of light scattered backwards by the human eye lens when illuminated by a laser diode. As our signal arises directly from the fundamental pathology-increased scattering in the lens-it directly assesses cataract extent and progression. Initial clinical results in undilated human eyes show device reading correlations in agreement with clinical examination and Scheimpflug photography.

Quantitative differentiation of dyes with overlapping one-photon spectra by femtosecond pulse shaping.

We demonstrate that DiI and Rhodamine B, which are not easily distinguishable to one-photon measurements, can be differentiated and in fact quantified in mixture via tailored two-photon excitation pulses found by a genetic algorithm (GA). A nearly three-fold difference in the ratio of two-photon fluorescence of the two dyes is achieved, without a drop in signal of the favored fluorophore. Implementing an acousto-optic interferometer, we were able to prove that the mechanism of discrimination is second-harmonic tuning by the phase-shaped pulses to the relative maxima and minima of these cross-sections.

Control of two-photon fluorescence of common dyes and conjugated dyes.

We present a comprehensive study of the selective excitation of two-photon fluorescence from various pairs of dyes and dyes in different conjugation states with tailored pulse shapes found with a genetic algorithm (GA). We investigate a number of biologically important dyes, and include dyes conjugated to trastuzumab (Herceptin(R)) and to a poly(amidoamine) dendrimer. We consider in detail the ability of tailored pulse shaping to discriminate dyes with significant spectral overlap.

Control of the blue fluorescent protein with advanced evolutionary pulse shaping.

We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal.

Physicochemical properties of blue fluorescent protein determined via molecular dynamics simulation.

Blue fluorescent protein (BFP) is a mutant of green fluorescent protein (GFP), where the chromophore has been modified to shift the emitted fluorescence into the blue spectral region. In this study, MD calculations were performed with the GROMACS simulation package and AMBER force field to investigate the dependence of BFPs physicochemical properties on temperature and applied pressure. The MD approach enabled us to calculate the compressibility of protein itself, separately from the nontrivial contribution of the hydration shell, which is difficult to achieve experimentally.

Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature.

Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore.

Vibrations-determined properties of green fluorescent protein.

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors.