Radiant energy – Luminophor irradiation
Reexamination Certificate
1999-09-14
2002-09-24
Epps, Georgia (Department: 2873)
Radiant energy
Luminophor irradiation
C356S318000
Reexamination Certificate
active
06455861
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to equipment and methods for assaying the amount of optical fluorescence, and the degree of fluorescence polarization, in samples.
2. Background and Description of the Related Art
Fluorescence Methods and Terminology.
Fluorescence involves exciting a molecular group with light of a first wavelength, causing it subsequently to emit light of a second, longer wavelength. The molecular group is termed a fluorophore, and the first and second types of light are termed “excitation” and “emission” light, respectively. Between excitation and emission, the molecular group is said to be in an excited state. Depending on the molecular group involved, the time spent in the excited state can vary widely, from a few nanoseconds to several microseconds. The duration of the excited state is termed the fluorescence lifetime. It is common to chemically engineer a fluorescent marker compound by grafting a fluorophore to a chemical group that reacts only or primarily with a very specific target molecule. The resultant fluorescent marker will bind only to very specific targets, and has fluorescence properties of the fluorophore.
Fluorescent markers are used to disclose the presence and/or location of targets within a sample, which may contain a variety of other compounds. For reliable detection, the other compounds in the sample must exhibit a very low degree of fluorescence, or there must be a way to discriminate between fluorescence emission resulting from the target compound and that from other compounds in the sample. Since the mechanism of fluorescence is present to at least some degree in most compounds, discrimination means are usually employed. Among the means are discrimination by wavelength of excitation light, by wavelength of emission light, and by fluorescence lifetime. Typically, discrimination by excitation wavelength involves making measurements using excitation light that has been filtered to contain light of only a selected wavelength band. Similarly, measurement of emission light through a filter that admits only a selected wavelength band provides a means to discriminate by emission wavelength.
Fluorescence lifetime discrimination is performed by a variety of methods, the simplest of which is to excite using a modulated source, and to observe emission using synchronous detection methods. For example, a target compound with a long fluorescence lifetime may be detected by exciting the sample with brief pulses, while measuring emission using a gated detector which is insensitive for a controlled, brief interval after excitation. Such a detector will not respond to compounds having a short fluorescence lifetime, which will have ceased emission by the time the detector becomes responsive. However, the target species, having a long fluorescence lifetime, will continue to emit for considerably longer, and the majority of its emission will be detected. Alternative approaches can also be used with single-element detectors, including detection with lock-in amplifiers, quadrature detection, and other standard signal analysis techniques. Imaging detection is possible with a gated intensifier or microchannel plate (MCP), by electronic shuttering, or by pixel shifting between photosensitive and non-photosensitive regions of a CCD detector.
Discrimination by these means is useful for removing so-called ‘background’ fluorescence arising from optical components, solvents, culture dishes, and the like, which are necessary elements in a fluorescence experiment but whose fluorescence signal is not seen as contributing meaningful information. That is, these are means for removing unwanted contaminant signals and for obtaining an enhanced signal-to-noise ratio in the sample fluorescence measurement.
It is possible to use discrimination techniques not just to remove background, but to learn more about the sample itself. For example, two or more fluorescent markers can be used which have distinct excitation or emission properties, so a single sample can be tagged with multiple markers to identify different structures or entities. The fluorescent signals are then resolved to obtain information about each marker independently. This technique is often termed multiprobe fluorescence.
Discrimination by excitation or emission wavelength may also be used to learn additional information about a single target species. For some fluorescent markers, the characteristic wavelengths of optimal excitation or emission vary with the chemical properties of the environment, such as the pH, salinity, concentration of calcium, or the presence of other, very specific molecules. Observing how fluorescence intensity varies with excitation wavelength, or measuring the spectrum of emission light, can provide a measurement of the chemical environment of the fluorescent marker. These practices are termed fluorescence excitation spectroscopy and fluorescence emission spectroscopy.
One case of special significance is fluorescence resonance energy transfer (FRET), which employs two molecular groups having carefully related properties. Typically, one group contains a fluorophore that is characteristically excited at a first wavelength and emits at a second wavelength. The other group is characteristically excited at the second wavelength and emits at a third wavelength. Depending on the presence, concentration, or molecular conformation of the two groups, the likelihood of interaction between the two groups is higher or lower. When the likelihood of interaction is high, energy is transferred from excited molecules in the first molecular group to the second molecular group, resulting in emission light at the third wavelength. Thus, sample emission at the third wavelength is enhanced and emission at the second wavelength is depressed, compared to the case when the likelihood of interaction is low. Typically, a FRET experiment involves excitation at the first wavelength while monitoring the level of emission at the second and third wavelength bands. From the ratio of emission at these bands, the level of interaction is inferred.
Additional information can be obtained in some cases by analyzing the fluorescence polarization (FP), which involves exciting the sample with linearly polarized light and measuring the degree of linear polarization in the emission light. Excitation light preferentially excites those molecules having a selected geometrical orientation relative to the light's polarization vector. Thus, the population of excited molecules is selectively oriented, rather than randomly so, at the time of excitation. If the fluorescence lifetime is comparable to or shorter than the molecular reorientation time, then the molecules will also be preferentially oriented when they emit, and the emission light will be linearly polarized to some degree. By measuring the degree of polarization, one infers the degree of preferential orientation at time of emission. It is conventional to refer to the degree of polarization (DOP) in terms of millipolarization units (MPU), defined as
DOP=1000*(
I
∥
−I
195
)/(
I
∥
+I
⊥
) [1]
where I
∥
and I
⊥
are the intensities of fluorescence emission polarized in the same sense as the excitation light and polarized orthogonal to it, respectively. Related measures are also used to quantify fluorescence polarization, based on substantially the same information.
Many factors can affect molecular re-orientation time, including rotational viscosity, temperature, and whether the fluorescent molecule is bound to another molecule or not. Measurement of fluorescence polarization (FP) is often used as a way to assess whether a molecule is in its bound or free state.
Equipment used in Fluorescence Instruments
It is common to use mercury or xenon arc lamps with optical filters for fluorescence excitation. These are very useful for providing ultraviolet (UV) light, which some fluorophores require. When only visible light is required for excitation, a less expensive tungsten
Cambridge Research & Instrumentation Inc.
Epps Georgia
Hanig Richard
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