We demonstrate a high speed method to analyze the impact of self-quenching on fluorescence lifetimes in individual cells by phase-sensitive flow cytometry, using a model system consisting of FITC (fluorescein isothiocyanate) labeled antimouse Thy 1.2 antibodies bound to murine thymus cells.

Experimental spectroscopic investigations of the absorption and luminescence properties of Er3+ ions doped into single crystals of the tetragonal-symmetry host LuPO4 are reported. The LuPO4 host permits the incorporation of relatively high concentrations of lanthanide dopants such as Er3+. The fluorescence decay of the 4I13/2–4I15/2 emission was found to be governed by a single exponential decay process when excited with a low-intensity pump source. However, experiments performed on samples of different concentrations show that Er3+ ions in LuPO4 experience concentration quenching as well as self-trapping. Using high-Er-concentration samples, emissions were also observed at visible wavelengths due to up-conversion processes.

A flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making frequency-domain, excited-state lifetime measurements on cells/chromosomes labeled with fluorescent probes, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence signals are processed by 1) low-pass filtering to obtain conventional FCM de-excited signals and 2) phase-sensitive detection electronics to resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase-shifts and to quantify fluorescence lifetimes in realtime. Processed signals are displayed as frequency distribution histograms and bivariate contour diagrams. Recent examples of biological applications include: 1) lifetime histograms recorded on autofluorescent human lung fibroblasts, murine thymus cells labeled with antibodies conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, and on cultured cells, nuclei, and chromosomes stained with DNA-binding fluorochromes and 2) phase-resolved, fluorescence signal-intensity histograms recorded on autofluorescent HLFs labeled with immunofluorescence markers and on murine thymus cells labeled with Red 613-antiThy 1.2 and propidium iodide (PI positive ''dead'' cells) to demonstrate the resolution of signals from highly overlapping emission spectra. This technology will increase the number of fluorescent markers usable in multilabeling studies and lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

A novel method is described for the measurement and analysis of fluorescence decays of individual cells and particles in flow. It combines the rapid measurement capabilities of a flow cytometer and the robust measurement and analysis procedures of time-domain fluorescence-lifetime spectroscopy. For excitation we use a cw laser that is pulse modulated by an electro-optic modulator. The characteristics and the repetition rate of the excitation pulses can be easily adjusted to accommodate fluorescence decays with a wide range of lifetimes.

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell-surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

In frequency-domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase-shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring Lifetimes at multiple frequencies. in this article we explore the requirements and experimental design considerations for making such measurements in flow, We report a phase-sensitive now cytometric method that allows one to probe the excited state-lifetimes of labeled cells by using multiple simultaneous modulation frequencies, Application of this method is demonstrated by measuring fluorescence Lifetimes of labeled cells at two frequencies simultaneously, using a continuous-wave, dual-frequency modulated excitation in now, The dual-frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell-by-cell basis in real time, Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level. (C) 1995 Wiley-Liss, Inc.

We have developed a method for fluorescence lifetime measurements in a flow cytometer based upon the amplitude demodulation of the fluorescence signals using digital data acquisition techniques. Amplitude demodulation is one of the two methods by which excited state lifetimes may be investigated in the frequency domain. The other method involves the phase-shift measurements. In frequency-domain measurement techniques, the amplitude-demodulation and phase-shift data serve mutually complementary roles to enhance the analytical capabilities of the measurements. The purpose of having amplitude demodulation measurement capability is to obtain information that supplements, rather than replaces, that obtained by the phase-shift method alone. Application of amplitude demodulation measurements has been widely explored in static, cuvette-based, frequency domain systems. However, due to time dependence of the amplitude of the modulated fluorescence signal in a flow cytometer, the amplitude demodulation measurements in flow turns out to be more complicated than similar measurements in a static system. The goal of the present work is to explore the problems involved in amplitude demodulation measurements in flow (using digital method), through detailed theoretical modeling and use the model to develop a practical method that can be incorporated into a flow cytometer to measure amplitude modulation lifetimes. We experimentally verify the amplitude demodulation measurement capability of this method using fluorescent microspheres. The experimental measurements show good agreement with static frequency-domain measurements on microspheres in bulk suspensions.

The spectroscopy and lasing properties of several Cr4+ doped crystals and glasses are presented. Included is a discussion of the intensity dependence of the broad band, Cr4+ absorption.

Spectroscopic data on the absorption and the fluorescence spectra and fluorescence lifetimes are presented for Cr4+:Y2SiO5, the newest Cr4+-doped laser crystal. The fluorescence measurements were conducted at temperatures ranging from 10 to 310 K, and the absorption spectra were measured at room temperature and 77 K. Spectroscopic analysis of the absorption and the emission spectra is performed on the basis of a distorted tetrahedral symmetry for the Cr4+ Site, namely C3v. The tetravalent chromium substitutes for Si4+ in this crystal, and there is no evidence for the presence of chromium in any other valence state. The Cr4+ site is characterized by strong crystal field parameters. We have tentatively assigned the sharp line at 1146 nm in the low-temperature fluorescence spectrum (excited by 1064-nm radiation) to the spin-forbidden singlet-to-triplet transition 1E-3A2, and the broad band with the peak at 1225 nm to the spin-allowed 3T2-3A2 transition. Excitation at 532 nm results in an additional type of emission in the near infrared. A similar dependence of the lasing wavelength on the pump wavelength was observed in the laser operation of the crystal at 77 K et al., in Advanced Solid-State Lasers, L. L. Chase and A. A. Pinto, eds., Vol. 13 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1992), pp. 28-30].

Laser action in Cr4+ doped yttrium-orthosilicate Cr:Y2SiO5 (Cr:YSO) cooled to 77 °K was demonstrated for the first time with 1064nm and 532nm pumping from a pulsed Nd:YAG laser and broadband (@ 840nm) pumping from a Q-switched Cr:LiSAF laser.

Spectroscopic studies of Cr4+ ions in different hosts are reported. Absorption and fluorescence spectra of Cr:Y2SiO5,the newest member of the Cr4+ doped laser crystals are presented. Low temperature luminescence data in the near infrared (NIR) has shown evidence of two types of emissions from both Cr:Y2SiO5 and Cr:Ca2Al2SiO7 in the 1200 nm band. There is also indication of a second unstable emisión center in Cr:Mg2SiO4 in the 1200 nm band.

Laser action in Cr4+ doped yttrium-orthosilicate Cr4+:Y2SiO5 cooled to 77 K was demonstrated for the first time with 1064 and 532 nm pumping from a pulsed Nd:YAG laser and broad band (@ 840 nm) pumping from a Q-switched Cr:LiSAF laser.