Photography – Plural image recording – Sequential recording on different areas of a single frame
Reexamination Certificate
1999-08-12
2001-03-27
Font, Frank G. (Department: 2877)
Photography
Plural image recording
Sequential recording on different areas of a single frame
C356S338000
Reexamination Certificate
active
06208815
ABSTRACT:
The present invention relates to a method for differentiating or detecting particles in a sample by identifying signal segments of time-resolved, optical raw signals from the sample on the basis of single photon detection according to claim
1
.
The method of fluorescence correlation spectroscopy (FCS) (WO 94/16313) and other confocal fluorescence techniques, as described in the Publication WO 96/13744 and in the European Patent Application 96 116 373.0, are particularly suitable for so-called homogeneous assays on a submicroliter scale. The latter application describes a method for analyzing samples by repeatedly measuring the number of photons per defined time interval in light which is emitted, scattered and/or reflected by the particles in the sample and determining the distribution of the number of photons in the respective time intervals, characterized in that the distribution of specific brightness of the particles is determined from the distribution of the numbers of photons. In these techniques, the actual measuring volume element is less than 10
−12
l. The term “homogeneous assays” means analytical methods in which all reacting components remain in one measuring compartment until the signal is detected. Excess components are not preliminarily separated from the reaction mixture, in contrast to the so-called ELISA methods. The above mentioned fluorescence techniques work particularly well if labeled reagents are employed in a range of between 1 pM and 0.1 &mgr;M. However, the kinetic boundary conditions of a chemical detecting reaction which is to proceed in the seconds to minutes range for practicability require the use of labeled detector reagents in excess over an analyte in the nanomolar range and above. This frequently means a great experimental difficulty in the performance of homogeneous assays if the concentration of the resulting reaction product is less than that of the excess reagent by one or more than one order of magnitude.
Homogeneous assays with confocal measuring techniques often do not offer any possibilities to keep the excess component out of the measuring volume with simple means. Only for oppositely charged particles, methods have been described (WO 94/16313) for separating excess components from the reaction product even within a reaction volume using electric fields. Unfortunately, however, such methods are unsuitable for many diagnostic detection methods of extraordinary economical importance. In particular, this applies to, e.g., the analytics of pharmacologically important receptors, e.g., on vesicles, the interaction of which with excess free labeled ligands is to be analyzed. Viruses, bacteria, cells, beads, particle fluids (e.g., with surface-bound specific chemicals as known from combinatorial chemistry), or just large molecular aggregates, such as ribosomes, can naturally be measured only in extremely low concentrations so that the use of excess reagent is unavoidable.
For homogeneous assay methods, there have been described ways to produce optical luminescence signals only in the case when complexes between the detector reagent and the particle to be detected have been formed, despite of the use of excess labeled detector reagent. These include, e.g., the methods of scintillation proximity assay (SPA), energy transfer assay through the formation of various donor-acceptor complexes or reaction products which revert luminescence quenching.
In confocal fluorimetry, the method of cross-correlation has proven useful. In cross-correlation methods, the detector reagent, e.g., bears marker A while the particle to be detected bears marker B. Although both the signals A′ from the detector reagent and B′ from the particle to be detected are recorded, in cross-correlation, only those signals are employed in which both signals A′ and B′ are recorded in the same time interval. This is the case when a complex of the detector reagent and the particle to be detected is present. In another variant, individual molecules of the detector reagent may be labeled with either of markers A* or B*. Those methods no longer yield satisfactory results when the concentration of the excess component is different from that of the complex to be detected by more than a factor of 10.
In various signal processing methods, there has been the problem of separating signals from the background noise. To solve it, the use of spectral filtering, lock-in and coincidence techniques, for example, has proven useful.
Spectral filtering is often easily done with optical signals. Thus, for example, in confocal microscopy or fluorescence correlation spectroscopy, the fluorescence signal is separated from the scattered light and the Raman emission from the solvent by using interference or colored glass filters. Wherever possible, the signal is resolved into a spectrum using a prism or grating. In many cases, however, this kind of filtering is unsufficient. For recording a complete spectrum, higher light intensities are necessary to obtain a minimum statistical base over the spectral range. Single particles, especially single molecules, are often not emitting enough photons, however. Although filters limit the spectrum to be measured, they require a detectable spectral difference.
The filtering of the signal after electric conversion into counts using fast Fourier transformation (FFT) requires assign-ability of the signal to frequencies. Correspondingly poor is the filtering performance of FFT and related techniques in analyzing the emission of particles the movement of which is random because of diffusion rather than regularly. Lock-in techniques also require a periodic signal as the base signal.
Coincidence techniques require a second signal as a trigger signal. Such a signal is hard to obtain from a particle or even a small molecule. If a pulsed laser is used, photons of scattered light appear in coincidence with the pulse of the light source, whereas luminescence photons mostly appear some nano-seconds later. This time difference is used, inter alia, for calculating the fluorescence lifetime. If a constant light source is used, however, as is mostly the case in FCS, there is no such possibility.
In Tellinghuisen et al. (“Analysis of Fluorescence Lifetime Data for Single Rhodamine Molecules in Flowing Sample Streams”, Analytical Chemistry 66, No. 1, 64-72, 1994), a method of fluorescence lifetime spectroscopy is described which serves to filter the photons derived from the light source, i.e., a laser in this case, from the overall signal. To achieve this, the signal counter current is compared with the trigger signal, i.e, the excitation pulse of the pulsed laser. If a photon arrives simultaneously with the excitation pulse, considering the velocity of light, it is identified as a scattered light pulse and deleted. One drawback of this method is that detector pulses which are not generated simultaneously with the excitation pulse are not recognized. Examples thereof include dark currents in the detector. In addition, fluorescence photons, if coinciding with the excitation pulse within the limits of time resolution of the trigger signal, are also deleted. This may lead to inacceptable distortions in the subsequent signal processing. When relaxation signals are measured, as with fluorescence lifetime, there is an additional drawback in that the strongest portion of the signal is deleted, resulting in a dramatic deterioration of the signal-to-noise ratio.
Keller et al. (“Single-Molecule Fluorescence Analysis in Solution”, Applied Spectroscopy 50, No. 7, 12A-32A, 1996) describes another method for determining the fluorescence lifetime. In this method, the time intervals between successive impulses arriving at the detector are determined by counting the number of trigger pulses, generated with 100 kHz, between two successive photons. These counts are stored in successive channels of a multichannel scaler (MCS). Then, this MCS signal is subjected to a temporal fast Fourier transformation (FFT) for smoothing. If, after FFT, at least 5 time intervals of the smoo
Gunther Rolf
Lupke Stefan
Seidel Claus
EVOTEC BioSystems AG
Font Frank G.
Jacobson Price Holman & Stern PLLC
Natividad Phi
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