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J R Alcala

Publications and source records attributed to J R Alcala.

8 recordsLinked to original sources
PubMed1997

Real-time frequency domain temperature and oxygen sensor with a single optical fiber.

The combined excited-state phosphorescence life-times of an alexandrite crystal and platinum tetraphenylporphyrin Pt(TPP) in a single-fiber sensor are used to monitor temperature and oxygen concentration in the physiological range from 15-45 degrees C and 0-50% O2 with precision of 0.24 degree C and 0.15% O2 and accuracy of 0.28 degree C and 0.2% O2. A 500-micron cubic alexandrite crystal bound to the distal end of a 750-micron-diameter optical fiber core and the Pt(TPP) coated circumferentially with a length of 1 cm from the end of the same fiber are excited with pulsed super-bright blue LED light. This apparatus uses a 125-kHz sampler for data acquisition and frequency domain methods for signal processing. The instrument amplifies both the dc and ac components of the photomultiplier output and band limits the signal to 20 kHz. The fundamental frequency of the excitation is set to 488.3 Hz and the highest harmonic used is the 35th. This bandlimited signal is sampled and averaged over a few hundred cycles in the time domain. The frequency domain representation of the data is obtained by employing fast Fourier transform algorithms. The phase delay and the modulation ratio of each sampled harmonic are then computed. At least four log-spaced harmonic phases or modulations are averaged before decoding the two lifetimes of temperature and oxygen phosphorescent sensors. A component of zero lifetime is introduced to account for the excitation backscatter leakage through optical interference filters seen by the photodetector. Linear and second-order empirical polynomials are employed to compute the temperatures and oxygen concentrations from the inverse lifetimes. In the situation of constant oxygen concentration, the lifetime of Pt(TPP) changes with temperature but can be compensated using the measured temperature lifetime. The system drift is 0.24 degree C for the temperature measurement and 0.59% for the oxygen concentration measurement over 30 h of continuous operation. The instrumentation and methods allow for 6-s update times and 90-s full-response times.

Algorithms↗
PubMed1996

Real time frequency domain fibreoptic temperature sensor using ruby crystals.

The excited state phosphorescence lifetime of ruby crystals is used to monitor temperature in the physiological range from 15 degrees to 45 degrees C with precision and accuracy less than 1 degree C, in real time. Precision of 0.1 degree C is attained with 3 min integration times. A 500 micron cubic ruby crystal bounded to the distal end of an optical fibre of similar core dimensions is excited with pulsed Ne-He laser light of about 9 microW average power. The instrument uses a sampler for data acquisition, and frequency domain methods for data fitting. The instrument amplifies the a.c. components of the detector output and band limits the signal to 800 Hz. The fundamental frequency of the excitation is set to 24.41 Hz to obtain 32 or less harmonics. This band-limited signal is sampled and averaged between 20 and 100 cycles to obtain temperature measurements in real time. The frequency domain representation of the data is obtained by employing fast Fourier transform algorithms. The phase delay and the modulation ratio, of each sampled harmonic, is then computed. Five to 32 values of the phase and modulation are averaged before computing the sensor lifetime. The technique is capable of measuring precise and accurate excited state lifetimes from subpicowatt luminescent signals in plastic optical fibres. A least squares fit yields the lifetimes of single exponentials. A component of zero lifetime is introduced to account for the backscatter excitation seen by the photodetector leaking through optical interference filters. The phosphorescence lifetimes measured reproducibly to about six parts in 1000, with a 2 s integration time, are used to monitor physiological temperature. Temperatures are computed employing empirical polynomials. The system drift is 3% over 5 h of continuous operation. The instrumentation and methods allow 2.7 s update times and 50 s full response times.

Biomedical Engineering↗
PubMed1995

Light transmittance as an index of cell volume in hippocampal slices: optical differences of interfaced and submerged positions.

Light transmittance (T) in the CA1 region of hippocampal slices was measured during exposure to media of various osmolarities to determine the utility of optical measurements as an index of changes in cell volume. In slices positioned at the gas-liquid interface, hypo-osmotic medium consistently produced a decrease in T and hyperosmotic medium produced an increase in T. The magnitude of deltaT was graded as a function of the strength of osmotic change. All changes in T were reversible upon return to isosmotic medium. In contrast, osmotically induced changes in T in submerged slices were consistently opposite in direction to those observed in slices at the interface. The magnitude and direction of deltaT could be altered by systematic variation of the level of the bathing medium within the same chamber, indicating that both extrinsic optical properties of various interfaces, such as refraction and reflection, and intrinsic optical properties of the tissue contribute to the observed T. Spectral measurements eliminated the possibility that osmotically induced deltaT was the result of changes in light absorbance by intrinsic chromophores such as cytochromes or hemoglobin. The results show that measurements of deltaT can be a useful index of changes in cell volume in brain slices, provided that the level of the bath remains constant.

Animals↗
PubMed1987

Resolvability of fluorescence lifetime distributions using phase fluorometry.

The analysis of the fluorescence decay using discrete exponential components assumes that a small number of species is present. In the absence of a definite kinetic model or when a large number of species is present, the exponential analysis underestimates the uncertainty of the recovered lifetime values. A different approach to determine the lifetime of a population of molecules is the use of probability density functions and lifetime distributions. Fluorescence decay data from continuous distributions of exponentially decaying components were generated. Different magnitudes of error were added to the data to simulate experimental conditions. The resolvability of the distributional model was studied by fitting the simulated data to one and two exponentials. The maximum width of symmetric distributions (uniform, gaussian, and lorentzian), which cannot be distinguished from single and double exponential fits for statistical errors of 1 and 0.1%, were determined. The width limits are determined by the statistical error of the data. It is also shown that, in the frequency domain, the discrete exponential analysis does not uniformly weights all the components of a distribution. This systematic error is less important when probability and distribution functions are used to recover the decay. Finally, it is shown that real lifetime distributions can be proved using multimodal probability density functions. In the companion paper that follows we propose a physical approach, which provides lifetime distribution functions for the tryptophan decay in proteins. In the third companion paper (Alcala, J.R., E. Gratton, and F.J. Prendergast, 1987, Biophys. J., in press) we use the distribution functions obtained to fit data from the fluorescence decay of single tryptophan proteins.

Fluorescence↗
PubMed1987

Fluorescence lifetime distributions in proteins.

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased. Finally, lifetime distributions for proteins interconverting between two conformations, each characterized by a quasi-continuum of energy substates, are presented. The origin of negative components of the lifetime distribution is also discussed. In the companion paper that will follow (Alcala, J. R., E. Gratton, and F. J.Prendergast, 1987, Biophys. J., in press) lifetime distributions obtained here are used to fit experimental data.

Mathematics↗
PubMed1987

Interpretation of fluorescence decays in proteins using continuous lifetime distributions.

The decay of the tryptophanyl emission in proteins is often complex due to the sensitivity of the tryptophan excited state to its surroundings. The traditional analysis of the decay curve using exponential components is based on the identification of each component with a particular protein conformation. An alternative approach assumes that proteins can exhibit a large number of conformations and that, at room temperature, the interconversion rate between conformations can be of the same order of magnitude as the excited-state decay rate. Following this assumption, the analysis of the protein emission was performed using continuous distributions of lifetime values. The number of average protein conformations, the range of mobility around each conformation, and the rate of interconversion between conformations determines the characteristics of the lifetime distribution. The fluorescence decay from some single tryptophan proteins was measured using multifrequency phase fluorometry and analyzed using a sum of exponentials, unimodal and bimodal probability-density functions, and the analytical form for lifetime distribution obtained for a model in which the tryptophan residue can move in a single potential well. For ribonuclease T1 and neurotoxin variant 3, the sum of two exponentials and bimodal probability-density functions gave comparable results, whereas for phospholipase A2, the description of the decay required three exponentials or bimodal probability-density functions. Also the temperature dependence of the fluorescence decay was investigated. It was found that the lifetime distribution was broader and shifted toward longer lifetime values at lower temperature. The analysis of the decay of tryptophan in buffer and of some tryptophan derivatives gave single-exponential decays. The single-potential well lifetime distribution, which has only three adjustable parameters, gave good fits for all cases investigated, but in the case of phopholipase A2, the temperature dependence of the parameters that describe the single-potential well distribution indicated the inadequacy of this model at lower temperature, suggesting that multiple potential wells can describe better the decay for this protein.

Kinetics↗