Radiant energy – Photocells; circuits and apparatus – Photocell controlled circuit
Reexamination Certificate
2002-09-30
2004-10-19
Le, Que T. (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Photocell controlled circuit
C250S573000
Reexamination Certificate
active
06806455
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and method for imaging time resolved fluorescence in biochemical and medical samples.
BACKGROUND OF THE INVENTION
Fluorescence is emitted when a fluorophore interacts with an incident photon (excitation). Absorption of the photon causes an electron in the fluorophore to rise from its ground state to a higher energy level. After a time dependent on the fluorophore and its environment, the electron reverts to its original level, releasing a photon (fluorescence emission) with wavelength dependant upon the amount of energy that is released during reversion. A fluorophore may emit at single or multiple wavelengths (creating an emission spectrum), as electrons drop from various orbitals to their ground states. The emission spectrum is constant for each species of fluorophore.
Fluorescent labels typically are small organic dye molecules, such as fluorescein, Texas Red, or rhodamine, which can be readily conjugated to probe molecules, such as steptavidin. The fluorophores can be detected by illumination with light of an appropriate excitation frequency and the resultant spectral emissions can be detected by electro-optical sensors or by eye.
Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modem Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Co, Inc. (1978), pp. 296-361 and the Molecular Probes Catalog (2001), OR, USA.
A fluorometer is an instrument that measures fluorescence. Fluorometers have three principal components: (a) light source for excitation, which is most typically a laser or broadband source; (b) filters and/or dispersive monochromators for selecting wavelength regions of interest, both in excitation and in emission; (c) a detector which converts the impinging fluorescence emission to an electrical signal. The present invention relates to fluorometers which measure fluorescence in biological samples, most specifically in biological samples used in drug discovery programs.
Some fluorometers (micro fluorometers) are constructed around confocal or noconfocal microscopes, and are designed for viewing fluorescently labeled cells or microvolumes of sample as discrete targets. There is a massive body of prior art relevant to such micro fluorometers. A subset body of this body of prior art relates to the use of micro fluorometers with microwell plates or wafers (e.g. Galbraith, et al., 1991; Rigler, 1995).
Other fluorometers (macro fluorometers) do not measure at cellular resolution but, rather, use low magnification optics to collect signal from each sample in a plurality of samples (as in U.S. Pat. No. 5,125,748 to Bjornson et al.; U.S. Pat. No. 5,340,747 to Eden), most typically wells are arranged within microwell plates (as in U.S. Pat. No. 6,071,748 to Modlin et al.; U.S. Pat. No. 5,670,113 to Akong et al.).
The means by which a plurality of samples is measured differs between a “scanning fluorometer”, a “stepping fluorometer” and an “area imaging fluorometer”.
In a scanning macro fluorometer (as disclosed in U.S. Pat. No. 5,672,880 to Kain), a plurality of samples is detected in serial fashion, by scanning an excitation beam over the sample and collecting each emission point serially. A scanning fluorometer can resolve each sample into multiple resolution points.
In a stepping macro fluorometer, the excitation beam and the detector (usually a photomultiplier tube or diode) move in stepwise fashion from sample to sample. The detector (or array of detectors) makes a unitary measurement from each sample, and does not discriminate multiple resolution points within each sample (as e.g. U.S. Pat. No. 5,589,351 to Harootunian; U.S. Pat. No. 5,670,113 to Akong et al.; U.S. Pat. No. 6,144,455 to Tuunanen et al; U.S. Pat. No. 6,127,133 to Akong et al.).
In an area imaging macro fluorometer, the detector is exposed to a plurality of samples in parallel (without scanning or stepping), and each sample is detected on a different portion of the detector area. By localizing different samples to different points on the detector, the area imaging fluorometer maintains the ability to discriminate discrete samples within a plurality of samples. The area imaging macro fluorometer may or may not discriminate multiple resolution points within each sample.
Area imaging macro fluorometers are well known (e.g. Haggart 1994; U.S. Pat. No. 6,140,653 to Che; U.S. Pat. No. 6,069,734 to Kawano et al.; many commercial systems). Most of these are suitable for flat samples such as electrophoresis gels. Some (e.g. U.S. Pat. No. 5,275,168 to Reintjes et al.; U.S. Pat. No. 5,309,912 to Knuttel) require highly specialized methods such as Raman spectroscopy or phase/amplitude modulation of illumination, and would not be suitable for either prompt or time resolved fluorescence intensity measurements as used with a plurality of biological samples. Others (U.S. Pat. No. 5,854,684 to Stabile et al.) mention the general use of imaging detectors to quantify light reflected from a plurality of samples, without describing specific embodiments within the illumination and optical detection systems that would allow effective use of the imaging detectors in macro fluorometry. Finally, others (WO96/05488 to McNeil et al.) disclose specific means to accomplish area imaging macro fluorometery, which means are not optimal for the purpose.
There is a need for high rates of sample throughput in drug discovery applications, wherein fluorescent specimens are usually arrayed within a plurality of wells and high throughput is required. While an area imaging fluorometer offers known advantages in its ability to quantify large numbers of samples in parallel, thereby achieving high throughput, specific embodiments appropriate to this purpose are required if the instrument is to be both high in throughput and high in sensitivity.
It is obvious, in light of the art of area imaging macro fluorometers, to position a CCD camera so as to image a specimen, deliver fluorescence excitation by known means, and collect emitted light using a lens in a known fashion. However, imaging fluorometers of the kind disclosed by McNeil et al. (WO96/05488) do not achieve equivalence in sensitivity with non-imaging fluorometers, and suffer from errors in signal detection. For example, they use standard lens systems which do not contain mechanisms so calculated as to minimize reflections and other forms of spurious signal, suffer from parallax error, and usually require that microwell plates be of the kind with a transparent bottom, through which the samples are read. In contrast, non-imaging fluorometers are highly sensitive, have no parallax error, are able to read from top or bottom of a plate, and do not require expensive transparent-bottom plates. Potential users of area imaging fluorometers in drug screening have not adopted widely any prior art system, because of factors such as those detailed above.
Challenges in applying area imaging fluorometry to drug screening arise, in the main, from four factors:
a) The wells form apertures which interact with the angle of incidence of the illumination. Wells which receive illumination at greater angles are less well illuminated than wells which receive more direct illumination. As most area imaging fluorometers deliver illumination from a region lateral to the collection lens (e.g. Neri et al., 1996), they can be successful with flat specimens but fail to properly illuminate deep wells. In practice, it is not uncommon to observe variations in excess of 300% in the delivery of light to wells lying at different positions in a microwell plate unless illumination delivery occurs only through the bottom of the microwell plate (as in U.S. Pat. N
Donders Paul
Masoumi Zahra
Ramm Peter
Yekta Ahmad
Zarate Carlos
Darby & Darby
Imaging Research Inc.
Le Que T.
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