MXPA01004566A - Sample analysis with successive quanta timing code - Google Patents

Abstract

In analyzing radiation (15) from a sample (14), single-quanta counting can be used to advantage especially at low levels of radiation energy, e.g., in the detection of fluorescent radiation. Preferred detection techniques include methods in which (i) fluorescence-stimulating radiation (12) is intensity-modulated in accordance with a preselected code, (ii) fluorescent radiation (15) is intensity-modulated with the preselected code, and/or (iii) modulation with the preselected code is applied to the sample (14) to influence a property which functionally affects emitted fluorescent radiation (15). The time intervals between signals (18) from a sensing element of a single-photon detector (17) are determined, recorded, and compared with the preselected code.

Description

SAMPLE ANALYSIS WITH SUCCESSIVE QUANTIC TIMING CODE Technical Field The invention relates to analytical technology and, more specifically, to the detection of a fluorescent species or fluorophore in a sample. Background of the Invention Fluorescent or fluorescent species emit fluorescent radiation when adequately stimulated by stimulation radiation. The emitted radiation can be used for chemical / biological analytical purposes, for example, to determine if a fluorophore of interest is present in a sample and to quantify its concentration. An analytical technique of this type is described in U.S. Pat. No. 5,171,534 to Smith et al., Wherein the DNA fragments produced in the DNA sequence are characterized based on the fluorescence of chromophores bound to the fragments. The stimulation electromagnetic radiation may be monochromatic or may include significant energy in a plurality of energy bands, for example, as described in US Pat. No. 5,784,157 to Gorfinkel et al. Stimulation radiation commonly varies in time, either randomly or regularly. The regular variation of the radiation intensity can be introduced artificially by modulating the intensity of the radiation source or the transmittance or reflectance of a filter element in the optical path. Regularly, regulated radiation can be determined as coded radiation if the temporal variation of the radiation is used as an information vehicle. Associated with such coded radiation is a temporal code, that is, a time domain function that corresponds to the temporal evolution of the intensity of the modulated radiation. A time domain function can be formed as a linear combination of several suitable functions whose respective contributions to the linear combination can be quantified reliably. Suitable in this respect are the sinusoidal time functions, for example, oscillating at different frequencies. In the prior art techniques, the coded radiation is considered continuous, with the time dependence of the detected radiation intensity considered as a continuous time domain function. The additional background includes several known single photon detection techniques for which W.R. McCluney, Introduction to Radiometry and - Photometry, (Introduction to Radiometry and Photometry) Artech House, 1996, pp. 114-122, provides a general introduction. Such techniques are designed to measure modulated radiation and can be classified into two groups: (a) asynchronous photon counting and (b) synchronous detection. As described in Alan Smith, Selected Papers in Photon Counting Detectors, (Selected Documents on Photon Count Detectors) SPIE, Vol. MS 413, 1998, asynchronous photon counting methods (a) involve the detection of a number of photons during a fixed interval of time, for example, a second, called the record interval. These methods allow the determination of an average arrival frequency of the photon. This frequency varies in time, either randomly or regularly and the synchronous count can be used to measure the variation of time. An essential limitation of this method is associated with the impossibility of measuring modulation frequencies greater than the repetition rate of the recording intervals. This difficulty is inherent in the principle of asynchronous counting, which is to keep track of the total number of photons received during the recording interval instead of recording their arrival times. A difficulty arises when the higher frequency fm? D in the modulation radiation modulation spectrum is comparable to or greater than the average fPhot frequency of the single photon detection. In this case, if the frequency limit is increased by reducing the time interval chosen for counting, the technique becomes increasingly inefficient since the counter will not count at all during most of the recording intervals. The methods (b) of synchronous detection involve the measurement of the arrival time of incident single photons. This time can refer to an "absolute" chronometer, or it can be measured in relation or "synchronously" with an excitation activating signal. The activating signal may be associated, for example, with the arrival of the first of the detected photons. Such methods are particularly valuable for the application in fast processes, for example, the fluorescent disintegration of a single coloring molecule excited as described, for example, by D.Y. Chen et al., "Single Molecule Detection in Capillary Electrophoresis; Molecular Shot Noise as a Fundamental Limit to Chemical Analysis "(" Simple Molecular Detection in Capillary Electrophoresis: Molecular Drive Noise as a Fundamental Limit for Chemical Analysis "), Analytical Chemistry, Vol. 68 (1996), page 690-696 , which typically requires special electronics to handle rapid temporal variations.An essential limitation of these methods is associated with the difficulty of maintaining records of high temporal resolution over a relatively long time.Thus, the detection of photon arrivals in the resolution The corresponding temporal time span of nanoseconds over a period of one second requires the acquisition of one trillion data records.This makes synchronous detection methods difficult to apply to the photometry of modulated single photon streams that vary relatively in shape. We have recognized that when detecting a species fl uorescent in a sample, the single photon count can be used to excel, especially at low fluorescent signal energy levels. Preferred detection techniques include methods in which (i) the fluorescence stimulation radiation is of intensity modulated according to a preselected code, (ii) wherein the intensity of the fluorescent radiation is modulated with the preselected code and (iii) ) wherein the modulation with a preselected code is applied to a sample to influence a property, for example, temperature, pressure or a force or frequency of the electric or magnetic field which functionally affects the emitted fluorescent radiation. Preferably, for the recording of the signals from a detection element of a single photon detector, the arrival time is recorded, optionally in conjunction with the recording of the time intervals. Advantageously, in the interest of minimizing the number of pulses lost due to the close temporal spacing of the pulses, activators D can be included for counting a set of circuits. Preferred techniques are generally applicable to single photon photometry coded by time or particle flow. These involve the measurement of time intervals between the single photon / particle arrivals combined with the data analysis that allows the decoding of the coded radiation, that is, the discrimination between the possible alternative codes and the quantification of different combinations of mixtures of the codes. The techniques provide that the time intervals between successive pulses are measured asynchronously, without requiring an external chronometer reference or a special trigger signal. These provide efficient measurement and decoding of the single photon encoded by time or particle fluxes. Brief Description of the Drawings Figure 1 is a schematic of a first preferred technique according to the invention, using a modulated light source. Figure 2 is a schematic of a second preferred technique according to the invention, using a dispersive element.

Figure 3 is a schematic of a third preferred technique according to the invention, involving the temporal coding of different spectral components of a fluorescent signal. Figure 4 is a schematic of a fourth preferred technique according to the invention, for recording time parameters of a stochastic pulse sequence of constant configuration or the like. Figure 5 is a schematic of a fifth preferred technique according to the invention, wherein the fourth technique is integrated with the measurement of time intervals. Figure 6 is a schematic of a sixth preferred technique according to the invention, wherein the fourth technique is argued for the additional minimization of pulses lost to the record. Detailed Description For the purposes of the present description, no distinction needs to be made between "photon" and "quantum", since each may result in a detector signal, typically in an electrical signal or pulse for electronic processing in accordance with the techniques of the invention. The use of other types of signal processing is not excluded, for example, by opto-electronic or purely optical means. It is understood that, in the alternative processing means, a detector signal or a pulse that is processed may be different from an electrical signal or pulse. A. Single Photon Detection in Methods for the Identification of Fluorophore A special lighting technique is used, with a plurality of modulated narrow band sources, each modulated according to its own distinguishable time domain function. The narrow-band sources excite different fluorophores differently, so that the emitted fluorescent radiation is encoded with information about the nature and composition of the illuminated fluorescent species. Photons are detected individually. In a first preferred embodiment as illustrated by Figure 1, a modulated multiple band light source that produces fluorescence excitation encoded radiation is combined with the single photon detection of the encoded fluorescence signal. Figure 1 shows the light source 11 producing a radiation flow 12 which, by means of an optical lighting system 13, is incident on the container 14 containing a fluorescent sample. The radiation flow 12 comprises a plurality of spectral bands, each modulated according to its own distinguishable time domain function. The fluorescent radiation 15 emitted by the fluorescent sample is received by an optical receiver system, for example, a target 16, and is directed towards the optical input of a single photon detector 17. The output of the detector 17 is a stochastic current 18 of electrical impulses of similar configuration, and the information about the intensity of the received fluorescent radiation in a reference time interval is contained in the average frequency of the pulses arriving in the interval. The temporal characteristics of the electric pulse current 18 are recorded in a suitable manner by the recorder 19 which is described in more detail below, in relation to Figures 4 and 5. In a preferred embodiment, the stochastic pulse current is characterized in terms of the time space between the arrivals of successive pulses. The detection system can be complemented by means of communication 120 to transfer the recorded information at an appropriate frequency from the recorder 19 to a signal processor unit 121. A second preferred embodiment as illustrated by Figure 2 can be viewed as a improvement over a known method for multicolored fluorescent detection, for example, as described in the above-referenced patent by Smith et al. In this technique, the fluorescent radiation emitted by an excited molecule is analyzed optically in different wavelength channels, for example, by means of a prism or a diffraction grating. The intensity of the fluorescent radiation in each of the wavelength channels is then determined by photometric means. In the second preferred embodiment, the sensitivity is increased by the use of single photon detection. Figure 2 shows the radiation 22 from a modulated optical source 21 focused by a lens 23 on a fluorescent sample 24. The modulated optical source 21 can produce one or more spectral bands that are modulated either together or independently with the domain functions of different time. The fluorescence emitted by the sample 24 in response to the incident radiation 22 is directed by an objective 26 to an optical processor comprising a dispersive element 27, for example a prism or a diffraction grating, and a set 29 of photon detectors. unique (SPD). The dispersive element 27 carries out the spectral analysis of the fluorescent signal. Each of the SPDs produces a stochastic current of simple electrical impulses at its output, and the information about the intensity of the received fluorescent radiation is contained in the temporal characteristics of the stochastic current with reference to Figure 2, the characteristics 210 - of each SPD are recorded by a registrar 211 whose structure will be described in more detail below in relation to Figures 4 and 5. In a preferred embodiment, also described in more detail below in relation to Figures 4 and 5, The description of the stochastic pulse current is specified in terms of the time separations between the successive pulse arrivals. The detection system further comprises a signal processor unit 212 and means for transferring the recorded information at a suitable frequency from the recorder 211 to the signal processor unit 212. Figure 2 illustrates a combination of a modulated light source for the excitation of fluorescence with a dispersive element to analyze the fluorescent response within different spectral bands, and the detection of single photon modulated fluorescence in each of the spectral bands. Additionally, as in Figure 1, the modulated light source can also be multi-band, so that the radiation flow 22 comprises a plurality of spectral bands, each modulated in accordance with its own distinctive time domain function. In this case, a preferred technique in which the different fluorescent species are distinguished both by their fluorescence emission spectrum and by their fluorescence excitation spectrum is additionally advantageous. This increases the fidelity of the fluorophore identification. A third preferred embodiment of the invention, illustrated by Figure 3, can be seen as an improvement over a technique known for multicolored fluorescent detection, for example, as applied in accordance with the above-referenced patent by Smith et al. The known technique is combined with single photon detection, using a modulation technique described in US Pat. No. 08 / 946,414, filed October 7, 1997 by Gorfinkel et al. According to this last technique, the radiation reflected, transmitted or fluorescently emitted by an object is coded in such a way that the coded radiation carries information about the properties of the object, for example, its color characterized by reflected wavelengths, or the identity and quantitative content of fluorescent species present in the object. In the present embodiment of the invention, the temporal coding of the different spectral components of a fluorescent signal is combined with the single photon detection of the coded spectral components, for improved sensitivity. Figure 3 shows the radiation 32 from the optical source 31 focused by a lens 33 on a fluorescent sample 34. In contrast to the modalities illustrated by Figures 1 and 2, the optical source 31 does not need to be modulated, and the radiation 32 may or may not be coded. The fluorescence emitted by the sample 34 in response to the incidental radiation 32 is directed by an objective 36 on an optical processor comprising a dispersive element 37, for example, a prism or a diffraction grating, and a set of optical modulators 38. The dispersive element 37 carries out the spectral analysis of the fluorescence 35. The spectral components are directed on a set of optical modulators 38 that modulate in time the spectral components solved in such a way that each different resolved spectral component is encoded by a different function of time. The modulated components 39 of the fluorescent spectrum are combined by an optical element 310 in an optical flow 311 focused on the optical input of the single photon detector 312. The output of the detector 312 represents a stochastic current 313 of electrical impulses of similar configuration, whose characteristics are recorded by means of the recorder 314 which is described in more detail below in relation to Figures 4 and 5. In a preferred embodiment, also described below in more detail, the description of the stochastic pulse current is specified in terms of the separation temporary between the arrivals of successive impulses. The detection system further comprises means 315 for transferring the recorded information at an appropriate frequency to a unit of the signal processor 316. B. Single Photon Detection of Modulated Photon Flows A fourth preferred embodiment of the invention is illustrated by FIG. invention, of a method for recording time parameters of a stochastic pulse sequence of constant configuration or the like. The recorder of Figure 4 operates with a controlled time resolution, controlled by a timer 45 which provides a regular sequence of electrical impulses of constant configuration defining the time intervals of the record. A stochastic current 41 of electrical input pulses can originate from a detection element of a single photon detector which is typically a photomultiplier tube (PMT) or an avalanche photodiode (APD). The input pulses do not need to be of the same configuration. With an APD, an avalanche extinction circuit is used either passively or actively. Typically, the APD is pre derived in its avalanche regime, so that the first photon initiates the avalanche. To prepare for the next arrival of the photon, the avalanche has to be extinguished. It may be advantageous to use the so-called forced extinction circuit that regularly extinguishes the condition of the avalanche, regardless of whether an avalanche has in fact been initiated, so that the arrival of photons and the extinction time do not correlate. As a result, the duration of the impulse avalanche will also be stochastic, depending on the time of arrival of the photon in relation to the subsequent extinction. The pulse current 41 is directed to a state-n cyclic state shift device or recorder 42. Such a device has successive stable n-states which can be numbered 0, 1, 2, ..., nl, with a change from a state k to its successor state k + 1, which are activated by an input pulse, and with a state nl that has the state 0 as its successor state. Between the input pulses, the n-state cyclic state moving device 42 retains its state. For example, a jogger having a sequence of stable states 0, 1, 0, 1, ... can be used for a 2-state cyclic state shift device, with each input pulse causing a transition from 0 to 1 or from 1 to 0. It is not necessary for the cyclic state shift device to return to its initial state when its status is read. This contrasts with conventional photon counters where each reading of counter data is accompanied by repositioning of the counter state back to the initial state.

- To be more specific, without limiting the invention, a jogger will be assumed in the following further description of Figure 4. The output from the jogger represents a stochastic sequence 43 of rectangular pulses of varying length. The sequence 43 is directed to a recording device 44, which may be perceived as an analog or digital signal recorder. The output signal 47 is transferred from the recording device 44 to a signal processor (not shown). The recorder of Figure 4 operates essentially asynchronously. But, in contrast to the asynchronous photon counters that record the total number of photons arriving in a particular time interval, the preferred recorder records their arrival times. The accuracy of the arrival time recording is controlled by the timer 45. The time intervals are recorded without measuring the duration of the intervals. This function can be carried out by one of several devices known to those skilled in the art, placed in an electrical circuit in a serial manner with the recorder and using its output signal 47. For example, a general purpose computer can be used to process the order of the data acquired by the recording device 44. In some applications it may be advantageous to integrate in a single device the functions of recording the time intervals between the successive single-photon detections and the measurements of the time intervals. Such a fifth integrated preferred embodiment of the invention is illustrated by Figure 5, for a stochastic electric pulse current 51 to which the configuration and extinction considerations of the APD relating to the pulses 41 of Figure 4 are also applicable. shown in Figure 5, a stochastic current of electrical pulses 51 is directed on a tilter 52. Its output represents a stochastic sequence 53 of rectangular input pulses of variable length. The sequence 53 slides in three directions between the counters 56 and 56 'and the delay line 531 controlled. The counter 56 receives the signal directly from the jogger, and the counter 56 'receives its signal through an inverter 521. In this way, the counters 56 and 56' are controlled by opposing phase signals. Instead of a tilter 52, a state-n cyclic state shift device can be used, as described with reference to Figure 4. Advantageously in this case, instead of two counters 56 and 56 ', more than one can be used. n counters A stopwatch 54 provides a regular sequence of electrical pulses of constant configuration that are counted by the counter 56. By way of example, the counter 56 is a counter whose input signals equal 1 to the chronometer pulse arrival time. Advantageously, if the pulses 51 originate from an APD, the external extinction circuit which periodically forces the APD out of its avalanche regime can be synchronized by the synchronizer 54. There is no advantage in increasing the extinction frequency beyond the frequency of the chronometer that provides the basic discretization of time in the technique. When a photon is detected and an electrical pulse 51 enters the jogger 52, one of the counters 56 and 56 'stops counting and the other starts counting. The counter that has just stopped counting then contains the register 57 of how long the interval between two successive pulses has lasted, measured in terms of the number of chronometer cycles counted. The register 57 is transferred to the registration device 510 via a switch 58 which serves to provide the successive recording at time intervals so that, while recording a time interval, the next one is measured. The switch 58 is controlled by a switching signal that is derived by the signals input 53 delayed by a characteristic time t corresponding to the response time of the counter 56. The output of the switch 58 represents a code sequence 59 describing the time intervals measured between the detected photons. The codes 59 appear in the output of the switch 58 stochastically corresponding to the detection of the photons entering and delayed by the time interval that is in the sum of T and the response time t2 of the switch itself. It is advantageous, consequently, controlling the recording device 510 by switching signals that are derived from the input signals 53, delayed from the time of switching the jogger by time t1 + t2. The output 514 of the recording device 510 represents the same sequence 59 of codes describing the time intervals measured between the detected photons. In contrast to the sequence 59 that accumulates in time stochastically, the sequence 514 may be transmitted in a regular manner, for example, at a regular frequency, for further processing. In addition to the technique illustrated by Figure 4, Figure 6 illustrates the inclusion of triggers D to minimize the number of uncounted pulses due to their close spacing in time. The electrical pulses of a single photon detector output are directed through a fast switch 61 to the input C of a synchronous 8-bit binary counter 62. The result of the count is passed to the storage register 63 as a word or byte of - 8 bits. To prevent the change of state of the counter 62 during storage, the synchronous pulse generator 65 shuts off the switch 61 simultaneously to send a short registration pulse to the input Wr of the storage register 63. The output of the storage register 63 travels through the 64 controller directly to the parallel port of a computer. The operational control error indicator is provided by a logic comparator 66 equipped with an LED (light emitting diode) 67. The parallel computer port is synchronized by a synchronous pulse through the delay line 68 with an appropriate delay t . The same delayed pulse synchronizes the logic comparator 66. For an exemplary embodiment of the technique illustrated by Figure 6, the following may be specified and perceived: a discretization frequency of 125 KHz, a maximum number of pulses per discretization interval of 256, a minimum time between recorded pulses of 20ns, a maximum average frequency of recorded pulses of 32 MHz, and a minimum fraction of lost photons of 0.25%. The techniques of the invention can be used to excel in a variety of applications involving electromagnetic coded radiation, including multicolored luminescent detection based on fluorescence spectroscopy and fluorescence excitation spectroscopy. These can be used in general sensor applications with other modulated luminescence signals such as those based on various spectroscopic techniques such as transmission, absorption, reflection, or Raman spectrum, as well as electroluminescence, chemiluminescence and the like. The techniques are especially useful for detecting weak signals, for example, those that prevail in optical communication links where signals are transmitted over long optical fibers.

Claims (26)

1. CLAIMS 1. A method for detecting a fluorescent species in a sample, comprising the steps of: irradiating the sample with stimulation radiation, to stimulate a fluorescent emission of the species, and with intensity modulated stimulation radiation over time of according to a preselected code; detect the successive quantum of the fluorescent emission; determine the time intervals between the quantum detection instances; record a sequence of time intervals; and compare the recorded sequence with the code.
2. 2. The method according to claim 1, wherein the stimulation radiation has energy in a single energy band.
3. The method according to claim 1, wherein the stimulation radiation comprises different spectral components, each of the components being modulated in intensity according to a different pre-selected code.
4. 4. The method according to claim 1, further comprising dispersing the fluorescent emission in different spectral components for separate detection.
5. 5. The method according to claim 1, wherein the time interval is measured directly.
6. The method according to claim 1, wherein the measurement of the time interval comprises sampling the state of a cyclic state shift register that changes its state each time a quantum is detected.
7. 7. A method for detecting a fluorescent species in a sample, comprising the steps of: irradiating the sample with stimulation radiation, to stimulate a fluorescent emission of the species; Modulate the fluorescent emission according to a preselected code; detect the successive quantum of the modulated fluorescent emission; determine the time intervals between the quantum detection instances; record a sequence of time intervals; and compare the recorded sequence with the code.
8. The method according to claim 7, wherein the stimulation radiation has energy in a single energy band.
9. The method according to claim 7, wherein the stimulation radiation comprises different spectral components, each of the components being modulated in intensity according to a different pre-selected code.
10. The method according to claim 7, further comprising dispersing the fluorescent emission in different spectral components for separate detection.
11. The method according to claim 7, wherein the time interval is measured directly.
12. The method according to claim 7, wherein the measurement of the time interval comprises sampling the state of a cyclic state shift register that changes its state each time a quantum is detected.
13. 13. A method for detecting a fluorescent species in a sample, comprising the steps of: irradiating the sample with stimulation radiation, to stimulate a fluorescent emission from the species; physically influencing the sample in a modulated form in accordance with a preselected code to correspondingly modulate the emission; detect the successive quantum of the modulated fluorescent emission; determine the time intervals between the quantum detection instances; record a sequence of time intervals; and compare the recorded sequence with the code.
14. The method according to claim 13, wherein the stimulation radiation has energy in a single energy band.
15. 15. The method according to claim 13, wherein the stimulation radiation comprises different spectral components, each of the components being modulated in intensity according to a different pre-selected code.
16. 16. The method according to claim 13, further comprising dispersing the fluorescent emission in different spectral components for separate detection.
17. 17. The method according to claim 13, where the time interval is measured directly.
18. The method according to claim 13, wherein the measurement of the time interval comprises sampling the state of a cyclic state shift register that changes its state each time a quantum is detected.
19. 19. A method for analyzing a sample, comprising: detecting the successive quantum of the modulated intensity radiation of the sample, the modulation being over time according to a preselected code; determine the time intervals between the - quantum detection instances; record a sequence of time intervals; and compare the recorded sequence with the code.
20. 20. The method according to claim 19, wherein the radiation from the sample is electromagnetic radiation.
21. 21. The method according to claim 19, wherein the radiation from the sample is particle radiation.
22. 22. The method according to claim 19, wherein the radiation has been stimulated by incident stimulation radiation on the sample.
23. 23. The method according to claim 22, wherein the intensity modulation of the radiation from the sample is due to the intensity modulation of the stimulation radiation.
24. The method according to claim 20, wherein the intensity modulation from the sample is due to physically influencing the sample in a modulated manner.
25. 25. Apparatus for analyzing a sample, comprising: a detector residue for detecting the successive quantum of the intensity radiation modulated from the sample, the modulation being on time according to a preselected code; a residue of time interval determination operably coupled to the detector residue to determine time intervals between the quantum detection instances; a recorder residue operably coupled to the time interval determination residue to record a sequence of the time intervals; and a comparator residue operably coupled to the recording residue to compare the recorded sequence with the code.
26. 26. Apparatus for making a sample, comprising: detector means for detecting the successive quantum of the intensity radiation modulated from the sample, the modulation being on time according to a preselected code; time slot determining means operably coupled to the detector means to determine time intervals between the quantum detection instances; recorder means operably coupled to the time interval determining means for recording a sequence of the time intervals; and comparator means operably coupled to the recorder means for comparing the recorded sequence with the code.