Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-01-22
2001-11-13
Font, Frank G. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S318000, C356S417000, C250S458100, C250S459100
Reexamination Certificate
active
06317207
ABSTRACT:
This application incorporates by reference the following U.S. patent applications: Ser. No. 09/062,472, filed Apr. 17, 1998; Ser. No. 09/160,533, filed Sep. 24, 1998; Ser. No. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/468,440, filed Dec. 21, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; and Ser. No. 09/494,407, filed Jan. 28, 2000.
This application also incorporates by reference the following PCT patent applications: Ser. No. PCT/US98/23095, filed Oct. 30, 1998; Ser. No. PCT/US99/01656, filed Jan. 25, 1999; Ser. No. PCT/US99/03678, filed Feb. 19, 1999; Ser. No. PCT/US99/08410, filed Apr. 16, 1999; Ser. No. PCT/US99/16057, filed Jul. 15, 1999; Ser. No. PCT/US99/16453, filed Jul. 21, 1999; Ser. No. PCT/US99/16621, filed Jul. 23, 1999; Ser. No. PCT/US99/16286, filed Jul. 26, 1999; Ser. No. PCT/US99/16287, filed Jul. 26, 1999; Ser. No. PCT/US99/24707, filed Oct. 19, 1999; Ser. No. PCT/US00/00895, filed Jan. 14, 2000; and Ser. No. PCT/US00/03589, filed Feb. 11, 2000.
This application also incorporates by reference the following U.S. provisional patent applications: Ser. No. 60/124,686, filed Mar. 16, 1999; Ser. No. 60/125,346, filed Mar. 19, 1999; Ser. No. 60/130,149, filed Apr. 20, 1999; Ser. No. 60/132,262, filed May 3, 1999; Ser. No. 60/132,263, filed May 3, 1999; Ser. No. 60/135,284, filed May 21, 1999; Ser. No. 60/138,311, filed Jun. 9, 1999; Ser. No. 60/138,438, filed Jun. 10, 1999; Ser. No. 60/138,737, filed Jun. 11, 1999; Ser. No. 60/138,893, filed Jun. 11, 1999; Ser. No. 60/142,721, filed Jul. 7, 1999; Ser. No. 60/153,251, filed Sep. 10, 1999; Ser. No. 60/164,633, filed Nov. 10, 1999; Ser. No. 60/167,301, filed Nov. 24, 1999; Ser. No. 60/167,463, filed Nov. 24, 1999; Ser. No. 60/178,026, filed Jan. 26, 2000; Ser. No. 60/182,036, filed Feb. 11, 2000; and Ser. No. 60/182,419, filed Feb. 14, 2000.
This application also incorporates by reference the following publications: Richard P. Haugland,
Handbook of Fluorescent Probes and Research Chemicals
(6
th
ed. 1996); and Joseph R. Lakowicz,
Principles of Fluorescence Spectroscopy
(2
nd
ed. 1999).
FIELD OF THE INVENTION
The invention relates to photoluminescence. More particularly, the invention relates to apparatus and methods for determining temporal properties of photoluminescent samples using frequency-domain photoluminescence measurements based on photon counting and/or the separation of measured luminescence into potentially overlapping time bins.
BACKGROUND OF THE INVENTION
Luminescence is the emission of light from excited electronic states of luminescent atoms or molecules (i.e., “luminophores”). Luminescence generally refers to all emission of light, except incandescence, and may include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, which includes fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In particular, the excited electronic state is created by the absorption of radiation having an energy sufficient to excite an electron from a low-energy ground state into a higher-energy excited state. The energy associated with the excited state subsequently may be lost through one or more of several mechanisms, including production of a photon through fluorescence, phosphorescence, or other mechanisms. Here, the terms luminescence and photoluminescence are used interchangeably, except where noted, and a reference to luminescence or luminophore should be understood to imply a reference to photoluminescence and photoluminophore, respectively.
Luminescence may be characterized by a number of parameters, including luminescence lifetime. The luminescence lifetime is the average time that a luminophore spends in the excited state prior to returning to the ground state.
Luminescence may be used in assays to study the properties and environment of luminescent analytes. The analyte may be the focus of the assay, or the analyte may act as a reporter to provide information about another material or target substance that is the focus of the assay. Luminescence assays may be based on various aspects of the luminescence, including its intensity, polarization, and lifetime, among others. Luminescence assays also may be based on time-independent (steady-state) and/or time-dependent (time-resolved) properties of the luminescence.
Time-resolved luminescence assays may be used to study the temporal properties of a sample. These temporal properties generally include any properties describing the time evolution of the sample or components of the sample. These properties include the time-dependent luminescence emission and time-dependent luminescence polarization (or, equivalently, anisotropy), among others. These properties also include coefficients for describing such properties, such as the luminescence lifetime and the rotational (or more generally the reorientational) correlation time.
Time-resolved luminescence may be measured using “time-domain” or “frequency-domain” techniques, each of which involves monitoring the time course of luminescence emission.
In a time-domain measurement, the time course of luminescence is monitored directly. Typically, a sample containing a luminescent analyte is illuminated using a narrow pulse of light, and the time dependence of the intensity of the resulting luminescence emission is observed. For a simple luminophore, the luminescence commonly follows a single-exponential decay, so that the luminescence lifetime can (in principle) be determined from the time required for the intensity to fall to 1/e of its initial value.
In a frequency-domain measurement, the time course of luminescence is monitored indirectly, in frequency space. Typically, the sample is illuminated using intensity-modulated incident light, where the modulation may be characterized by a characteristic time, such as a period. Frequency-domain analysis may use almost any modulation profile. However, because virtually any modulation profile can be expressed as a sum of sinusoidal components using Fourier analysis, frequency-domain analysis may be understood by studying the relationship between excitation and emission for sinusoidal modulation.
FIG. 1
shows the relationship between excitation and emission in a frequency-domain experiment, where the excitation light is modulated sinusoidally at a single modulation frequency f. The resulting luminescence emission is modulated at the same frequency as the excitation light. However, the intensity of the emission will lag the intensity of the excitation by a phase angle (phase) &phgr; and will be demodulated by a demodulation factor (modulation) M. Specifically, the phase &phgr; is the phase difference between the excitation and emission, and the modulation M is the ratio of the AC amplitude to the DC offset for the emission, relative to the ratio of the AC amplitude to the DC offset for the excitation. The phase and modulation are related to the luminescence lifetime &tgr; by the following equations:
&ohgr;&tgr;=tan(&phgr;) (1)
ωτ
=
1
M
2
-
1
(
2
)
Here, &ohgr; is the angular modulation frequency, which equals 2&pgr; times the modulation frequency. Significantly, unlike in time-domain measurements, the measured quantities (phase and modulation) re directly related to the luminescence lifetime. For maximum sensitivity, the angular modulation frequency should be roughly the inverse of the luminescence lifetime. Typical luminescence lifetimes vary from less than about 1 nanosecond to greater than about 10 milliseconds. Therefore, instruments for measuring luminescence lifetimes should be able to cover modulation frequencies from less than about 20 Hz to greater than about 200 MHz.
A similar approach may be used to study other temporal properties of a luminescent sample, such as time-resolved luminescence polarization, which may be characterized by a rotational (or more generally a reorientational) correlation time. The use of standard frequency-domain techniques to study such properties is described in the above-identified patent appli
French Todd E.
Modlin Douglas N.
Stumbo David P.
Font Frank G.
Kolisch Hartwell Dickinson & McCormack & Heuser
Lauchman Layla
LJL Biosystems Inc.
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