The duty cycle problem referred to by Alcala has been discussed in our original manuscript, and we calculated the reduction of the signal quite explicitly. His comments on the harmonic content are in error, and the correct values were given in the original paper.

The analog electronics commonly found in frequency domain fluorometers are limited to collecting only a single frequency at a time, and can be a source of systematic errors. We have developed an instrument in which most of the analog electronics are replaced with a computer-controlled digital-acquisition system. The computer is used for the direct collection of multifrequency data; filtering and calculation of the phase and modulation ratio are done by the software. From these values, fluorescence lifetimes can be determined. This new approach reduces most of the systematic errors due to the analog electronics' hardware and allows for reconfiguration of the instrument with only minor changes of the software. This digital-acquisition system is not a simple substitution of an analog element, but it provides a new function and new capabilities for frequency domain fluorometers. We have used this digital approach to build a "parallel" phase fluorometer which simultaneously collects and processes several modulation frequencies.

The fluorescence and polarization anisotropy decays of perylene in viscous solvents are investigated at several temperatures between -20 and 35 °C by using the technique of multifrequency phase and modulation fluorometry. The anisotropy decay data are globally analyzed over all temperatures studied and fit directly to physical quantities by using a compartmental model. We present a generalized compartmental model that can be used to calculate anisotropy decay arising from any type of interconversion that introduces a change in the fluorescence polarization. This generalized model provides a simple method to calculate the anisotropy decay for systems undergoing different kinds of interconversions such as excited-state reactions, rotational diffusion, "jump" motions, or any combination which can be treated in a compartmental form. The basic idea is to consider a rotation as an interconversion between compartments which represent different geometries with respect to the laboratory frame. Compartments due to other processes can be added to these rotational compartments. A matrix is constructed by using the decay rates of each compartment and the rates of interconversion between these compartments. The polarization of each compartment and initial conditions are contained in vectors. Using this formalism, the fluorescence anisotropy decay can be represented as a standard eigenvector-eigenvalue problem which may be solved analytically or numerically. As a simple example, this compartmental model is applied to the perylene molecule undergoing only rotational diffusional motions, and is used to globally fit the anisotropy decay data at various temperatures and viscosities. We fit directly to the physical quantities needed to define the compartments, using only three fitting parameters, two rotational diffusion constants, and the average angle between the absorption and emission dipoles. In previous studies, four parameters were used to fit the anisotropy decay of perylene, that is, two rotational correlation times and two preexponentials. The results of this study are shown to be in agreement with previous measurements. © 1989 American Chemical Society.

Multifrequency-phase and modulation fluorometry allows for accurate analysis of fluorescence decay in the frequency domain. Essential to these frequency domain methods is a high-frequency modulation of the light source. Techniques for generating wide-band modulation of light are currently limited to the use of Pockel's cells and intrinsically modulated sources such as mode-locked lasers and synchrotron radiation. We present a method that employs two acousto-optic modulators in series for use with cw light sources. This modulator system gives two orders of magnitude more intensity output than the Pockel's cell modulator and requires less than one-tenth of the rf driving power. In addition, the Pockel's cell system is limited to modulation frequencies less than 250 MHz, whereas the particular implementation discussed here gives a quasicontinuous distribution of modulation frequencies from dc to 320 MHz. To obtain this range of frequencies, acoustic standing waves are set up simultaneously in each modulator, and the desired modulation frequency is achieved by choosing the proper combination of the two standing-wave frequencies. Light modulation is obtained at twice each of the individual standing-wave frequencies and at the sum and difference of twice the two acoustic frequencies. Data are presented to illustrate the use of this system for the measurement of phosphorescence as well as fluorescence decay.

Nicotinamide adenine dinucleotide (NAD+) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and WAT) NAD+ metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD+ biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT and WAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of β-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD+-SIRT1-caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD+ synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD+ biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.