Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data

Although rapid-scan FT-IR has made many important advances in IR spectroscopy possible, it characterized by limitations that can interfere with some types of spectroscopic measurements. The first drawback is the wide modulation frequency range across the spectrum. This results since in the rapid-scan mode, modulation frequency is a function of both mirror speed and the wavelength of the IR light. Thus, difficulties may occur when studying systems involving detectors of specific, narrow bandwidths. Also, the depth profiling of photoacoustic samples can be obscured by this characteristic. A second limitation of the rapid-scan method lies in acquiring scans with sub-millisecond time resolution, since the fastest scanner can collect only about 50 scans per second at reasonable resolution. An alternative step-scan technique has been developed to deal with the above demanding applications. In this system, the moving mirror of the interferometer is "stepped" to precise locations where (using either amplitude or phase modulation) interferogram points are collected by means of lock-in amplifier. After acquisition of each interferogram point, the moving mirror is translated to the next optical retardation position. This system offers numerous advantages: Signal modulation is tunable and the same across the spectrum. This characteristic offers strong advantages in studies involving slow thermal detectors, such as photoacoustic and photothermal beam deflection spectroscopies. For repeatable events, time resolution is limited only by the speed of the acquisition electronics and detectors. Interferograms of short-lived states are collected by triggering the event of interest at each of the moving mirror's optical retardation positions and then following the response of the detector as a function of time. In this way, interferograms with time resolution of 5 microseconds or less can be collected. Finally, the step-scan system is fully compatible with a rapid-scan system, so that the user may easily switch from one mode to the other. Examples of step-scan applications will be presented and compared with corresponding spectra taken using rapid-scan methods.

Auditory detection of tone modulation has been modeled as envelope-peak detection of the cochlear filterbank responses [J. L. Goldstein, J. Acoust. Soc. Am. 41, 458–479 (1967)]. To study the ability of this model to quantify temporal smoothing in detection, new psychophysical experiments using a 4IFC paradigm were conducted, with the authors as subjects. AM or quasi-FM sinusoidally modulated tones were discriminated from the carrier tone. Only one should was modulated among the four pushed sounds in the paradigm. The first series of exploratory experiments (1994) focused on low modulation frequencies (4–16 Hz). A second series of systematic experiments (1996) with a wide range of modulation frequencies allowed estimation of detector smoothing at carrier frequencies of 0.25, 1, and 4 kHz. Despite differences between subjects in estimated spectral filtering, modulation thresholds for both subjects gave similar estimates of smoothing at each carrier frequency. At modulation frequencies above 20 Hz, the smoothing function showed similar dependence on carrier frequencies as found from the early experiments (1967). Below 20 Hz, the early experiments, which used a method of adjustment paradigm, reveal a hyperacuity. This suggests a second slower detection mechanism that exploits repeated presentations of test sounds. [Work supported by NSF Grant IBN-9728383.]

At a previous meeting of the society, Rickards and Clark [J. Acoust. Soc. Am. Suppl. 1 72, S54(1982)] showed that steady‐state evoked potentials could be recorded from alert humans in response to amplitude‐modulated tones over a wide range of modulation frequencies. A subsequent paper has shown that, while there is an amplitude response maximum for alert subjects at a modulation rate of approximately 40 Hz and this frequency is thus appropriate for optimum threshold detection, different circumstances apply for sleeping subjects. Sleeping subjects differ from alert subjects in both amplitude response and background EEG noise, as functions of modulation frequency. A detection efficiency function reveals that during sleep the optimum modulation frequency, dependent on stimulus carrier frequency, lies well above 40 Hz. For lower carrier frequencies a peak occurs at about 90 Hz, while at higher frequencies the optimum modulation frequency is as high as 150 Hz. These higher modulation rates allow more efficient detection than does 40 Hz. Stimuli with high modulation rates have been used successfully with sleeping subjects including neonates.

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH₂ provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

An important property of spin-torque nano-oscillators (STNOs) is their ability to produce a frequency modulated (FM) signal, which is very critical for communication applications. We here demonstrate a novel single sideband (SSB) modulation phenomenon using a magnetic tunnel junction (MTJ)-based STNO, which saves transmission bandwidth and in principle should minimize attenuation for wireless communication. Experimentally, lower single sidebands (LSSBs) have been successfully demonstrated over a wide range of modulation frequency, fm = 150 MHz-1 GHz. The observed LSSBs are determined by the intrinsic properties of the device, which can be modeled well by a nonlinear frequency and amplitude modulation formulation and reproduced in macrospin simulations. Moreover, our macrospin simulation results show that the range of modulation current and modulation frequency for generating SSBs can be controlled by the field-like torque and biasing conditions.

Substantial progress in the field of light spectroscopy and imaging of tissues was achieved when the group of Chance, Patterson and Wilson showed that the optical parameters of a turbid medium can be obtained from time resolved measurements of short light pulses propagating in the medium (Patterson et al, 1991a). Essentially, a fit of the intensity as a function of time, measured at some distance from the source, can provide separately the values of the absorption and of the reduced scattering coefficients. This demonstration was important because the focus was shifted from attempts to separate the scattering from absorption, using empirical corrections to the Beer-Lambert law, to a rigorous application of a physical model. During the same period, our lab proposed employing the Fourier transform equivalent concept using an intensity modulated light source (Gratton et al, 1990). Since frequency domain methods have better resolution and sensitivity and are much faster than the time domain methods, our proposal was followed by many others including Chance, Patterson and Wilson (Boas et al, 1993, 1994; Cui and Ostrander, 1993; Duncan et al, 1993; Kaltenbach and Kaschke, 1993; O’Leary et al, 1992; Patterson et al, 1991b; Tromberg et al, 1993). It is well known that time domain and frequency domain measurements are mathematically equivalent, when the frequency domain measurement is carried out using a wide range of modulation frequencies (Gratton et al, 1983; Alcala et al, 1984). However, frequency domain measurements can be made at a single modulation frequency, thereby sacrificing some of the information. The advantage of measurements at a single frequency is that they can be very fast, accurate and have an excellent signal-to-noise ratio.

The effect of stimulus envelope shape on the auditory steady-state response

We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.

A self-excited dust acoustic wave is synchronized by sinusoidal modulation of the ion density, and the wave is imaged by planar laser-light scattering. Space-time diagrams based on the images reveal how the nonlinearity of the plasma’s response causes the wave’s frequency to be synchronized to a multiple of 0.5, 1, 2, or 3 of the modulation frequency. Space-time diagrams also reveal wave front merging as is observed for a wide range of modulation frequencies.

This paper reports a modification of an interferometric technique [H. Tsuchida et al., "A Novel Technique for Measuring the Frequency Deviation of Semiconductor Lasers under Direct Modulation," Jpn. J. Appl. Phys. 22, L19-L21 (1983)] for measuring the frequency deviation and phase difference between the AM and FM modulation of semiconductor laser diodes. The frequency deviation can be determined by the fringe visibility of interference patterns provided the phase difference, AM index, and time delay in an unbalanced Michelson interferometer are given. It covers a wide range of modulation frequencies, i.e., 10 Hz to 10 MHz. This method has such advantages that a high speed photodetector is not necessary and the accuracy of the measurement is not reduced by the spectral linewidth of the lasers.

On the frequency correction in temperature-modulated differential scanning calorimetry of the glass transition

Brillouin optical correlation domain analysis (BOCDA) is an established method for the measurement of Brillouin frequency distribution, where direct modulation of a distributed feedback laser diode (DFB-LD) is usually adopted to implement a frequency-modulated light source. Recently, we reported that a phase delay occurring between the amplitude and frequency variations, called AM-FM phase delay, in the output of the modulated DFB-LD can cause considerable errors in the Brillouin frequency measured by a BOCDA system. The error induced by the AM-FM phase delay is systematic since it cannot be suppressed by averaging process while constantly distorts the distribution map of Brillouin frequency. In this paper, we demonstrate the suppression of the adverse effects of the AM-FM phase delay using an injection-locked DFB-LD as a light source of BOCDA system. Theoretical explanation on the origin of the systematic error in the BOCDA measurement is provided by simulations. The investigation on the modulation characteristics of the master and slave LD's in the injection-locking is performed, the result of which shows that the injection locking not only reduces the depth of amplitude modulation, but also keeps the phase delay of near 180° in a wide range of modulation frequency. In test measurements, the systematic errors are reduced by more than 70% on average using the slave LD as a common light source of the BOCDA system.

The use of fluorescence detected circular dichroism (FDCD) for monitoring the quenching of chiral molecules is described. This technique combines the specificity for chiral centers of circular dichroism (CD), the sensitivity and selectivity of the fluorescence measurement with the additional chemical information provided by fluorescence quenching. Spectra are acquired using a multidimensional FDCD spectrometer which obtains a FDCD matrix of spectral data as a function of multiple excitation wavelengths (CD information) and multiple fluorescence emission wavelengths. This combination of FDCD and fluorescence quenching produces a very selective monitor for chiral fluorophores.

Fluorescence Lifetime Spectroscopy in Multiply Scattering Media with Dyes Exhibiting Multiexponential Decay Kinetics

We seek a sufficient condition on initial data in order that the solution of Schrodinger equation has both analytically smoothing effect and exponential decay in time.