Abstract:
In analyzing radiation from a sample, 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 is intensity-modulated in accordance with a preselected code, (ii) wherein it is the fluorescent radiation which is intensity-modulated with the preselected code, and (iii) wherein modulation with a preselected code is applied to a sample to influence a property which functionally affects emitted fluorescent radiation. For registration of the signals from a sensing element of a single-photon detector, time of arrival is recorded, optionally in conjunction with registration of time intervals. Advantageously, in the interest of minimizing the number of pulses missed due to close temporal spacing of pulses, D-triggers can be included in counting circuitry.

Description:
TECHNICAL FIELD 
   The invention is concerned with analytical technology and, more specifically, with the detection of a fluorescent species or fluorophore in a sample. 
   BACKGROUND OF THE INVENTION 
   Fluorescent species or fluorophores emit fluorescent radiation when suitably stimulated by stimulating radiation. The emitted radiation can be used for chemical/biological analytic purposes, e.g. in determining whether a fluorophore of interest is present in a sample and in quantifying its concentration. One analytic technique of this type is disclosed in U.S. Pat. No. 5,171,534 to Smith et al. wherein DNA fragments produced in DNA sequencing are characterized on the basis of fluorescence of chromophores tagged to the fragments. Stimulating electromagnetic radiation may be monochromatic, or may include significant energy in a plurality of energy bands, e.g. as disclosed in U.S. Pat. No. 5,784,157 to Gorfinkel et al. 
   The stimulating radiation usually varies in time, either stochastically or regularly. 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 modulated radiation may be termed as encoded radiation if the temporal variation of the radiation is used as a carrier of information. Associated with such encoded radiation is a temporal code, i.e. a time-domain function which corresponds to the temporal evolution of the intensity of 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 sinusoidal functions of time, for example, oscillating at distinct frequencies. 
   In prior-art techniques, the encoded radiation is considered as continuous, with the time dependence of detected radiation intensity regarded as a continuous time-domain function. 
   Further background includes several known single-photon detection techniques for which W. R. McCluney,  Introduction to Radiometry and Photometry , Artech House, 1996, pp. 114-122 provides a general introduction. Such techniques are designed for measuring modulated radiation, and they can be classified into two groups: (a) asynchronous photon counting and (b) synchronous detection. As described in Alan Smith,  Selected Papers on Photon Counting Detectors , SPIE, Vol. MS 413, 1998, methods (a) of asynchronous photon counting involve the detection of a number of photons during a fixed time interval, e.g. one second, called the registration interval. These methods allow the determination of an average frequency of photon arrival. This frequency varies in time, either stochastically or regularly, and synchronous counting can be employed to measure the time variation. An essential limitation of this method is associated with the impossibility of measuring frequencies of modulation that are higher than the repetition rate of registration 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 registration interval rather than register their times of arrival. A difficulty arises when the highest frequency f mod  in the modulation spectrum of modulation radiation is comparable to or higher than the average frequency f phot  of 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 because the counter will count nothing during most registration intervals. 
   Methods (b) of synchronous detection involve measurement of the time of arrival of incident single photons. This time may be referenced to an “absolute” clock, or may be measured relative to or “synchronously with” a triggering excitation signal. The triggering signal may be associated with the arrival of the first of detected photons, for example. Such methods are particularly valuable for application to fast processes, e.g. the fluorescent decay of a single excited dye molecule as described, e.g., by D. Y. Chen et al., “Single Molecule Detection in Capillary Electrophoresis: Molecular Shot Noise as a Fundamental Limit to Chemical Analysis”,  Analytical Chemistry , Vol. 68 (1996), pp. 690-696, typically requiring special electronics for handling fast temporal variations. An essential limitation of these methods is associated with the difficulty of maintaining records of high temporal resolution for a relatively long time. Thus, detecting photon arrivals at the temporal resolution corresponding to nanosecond time intervals over a one-second period requires acquisition of a billion data records. This makes methods of synchronous detection difficult to apply to the photometry of relatively slowly varying modulated single-photon fluxes. 
   SUMMARY OF THE INVENTION 
   We have recognized that, in detecting a fluorescent species in a sample, single-photon counting can be used to advantage, especially at low levels of fluorescent signal energy. Preferred detection techniques include methods in which (i) fluorescence-stimulating radiation is intensity-modulated in accordance with a preselected code, (ii) wherein it is the fluorescent radiation which is intensity-modulated with the preselected code, and (iii) wherein modulation with a preselected code is applied to a sample to influence a property, e.g. temperature, pressure, or an electric or magnetic field strength or frequency which functionally affects emitted fluorescent radiation. 
   Preferably, for registration of the signals from a sensing element of a single-photon detector, time of arrival is recorded, optionally in conjunction with registration of time intervals. Advantageously, in the interest of minimizing the number of pulses missed due to close temporal spacing of pulses, D-triggers can be included in counting circuitry. 
   The preferred techniques are generally applicable to photometry of time-encoded single-photon or particle fluxes. They involve measurement of time intervals between single-photon/particle arrivals combined with data analysis that permits decoding of the encoded radiation, i.e., discrimination between alternative possible codes and quantification of different combinations of mixtures of the codes. The techniques provide for the time intervals between successive pulses to be measured asynchronously, without requiring an external clock reference or special triggering signal. They provide for efficient measurement and decoding of time-encoded single-photon or particle fluxes. 

   
     BRIEF DESCRIPTION OF THE DRAWING 
       FIG. 1  is a schematic of a preferred first technique in accordance with the invention, using a modulated light source. 
       FIG. 2  is a schematic of a preferred second technique in accordance with the invention, using a dispersive element. 
       FIG. 3  is a schematic of a preferred third technique in accordance with the invention, involving temporal encoding of different spectral components of a fluorescent signal. 
       FIG. 4  is a schematic of a preferred fourth technique in accordance with the invention, for registration of temporal parameters of a stochastic sequence of pulses of constant or similar shape. 
       FIG. 5  is a schematic of a preferred fifth technique in accordance with the invention, wherein the fourth technique is integrated with the measurement of time intervals. 
       FIG. 6  is a schematic of a preferred sixth technique in accordance with the invention, wherein the fourth technique is augmented for further minimization of pulses lost to registration. 
   

   DETAILED DESCRIPTION 
   For purposes of the present description, no distinction need be made between “photon” and “quantum”, as each can result in a detector signal, typically an electrical signal or pulse for electronic processing in accordance with techniques of the invention. Use of other types of signal processing is not precluded, e.g. by opto-electronic or purely optical means. It is understood that, in alternative processing means, a detector signal or a pulse being processed can be other than an electric signal or pulse. 
   A. Single-Photon Detection in Methods for Fluorophore Identification 
   A special illumination 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 illuminated fluorescent species. Photons are detected individually. 
   In a preferred first embodiment as illustrated by  FIG. 1 , a modulated multi-band light source producing encoded radiation of excitation of fluorescence is combined with single-photon detection of encoded fluorescence signal. 
     FIG. 1  shows the light source  11  producing a radiation flux  12  which, via an optical illumination system  13 , is incident on the container  14  holding a fluorescent sample. The radiation flux  12  comprises a plurality of spectral bands, each modulated according to its own distinguishable time-domain function. Fluorescent radiation  15  emitted by the fluorescent sample is received by an optical receiver system, e.g. an objective  16 , and is directed to the optical input of a single-photon detector  17 . The output of the detector  17  is a stochastic stream  18  of electric pulses of similar shape, and information about the intensity of the received fluorescent radiation in a set time interval is contained in the average frequency of the pulses arriving in the interval. The temporal characteristics of the stream  18  of electric pulses are registered in a suitable form by the recorder  19  which is described below in further detail, in connection with  FIGS. 4 and 5 . In a preferred embodiment, the stochastic stream of pulses is characterized in terms of the spacing in time between arrivals of successive pulses. The detection system may be complemented by communication means  120  for transferring the recorded information at an appropriate rate from the recorder  19  to a signal processor unit  121 . 
   A preferred second embodiment as illustrated by  FIG. 2  can be viewed as an improvement over a known method for multicolor fluorescent detection, e.g. as disclosed in the above-referenced patent to Smith et al. In this technique, the fluorescent radiation emitted by an excited molecule is optically analyzed into distinct wavelength channels, e.g. by a prism or a diffraction grating. The intensity of fluorescent radiation in each of the wavelength channels is then determined by photometric means. In the preferred second embodiment, sensitivity is enhanced by the use of single-photon detection. 
     FIG. 2  shows radiation  22  from a modulated optical source  21  being focused by a lens  23  onto a fluorescent sample  24 . The modulated optical source  21  may produce one or several spectral bands that are modulated either together or independently with distinct time domain functions. Fluorescence  25  emitted by the sample  24  in response to the incident radiation  22  is directed by an objective  26  to an optical processor which comprises a dispersive element  27 , e.g. a prism or a diffraction grating, and a set  29  of single photon detectors (SPD). The dispersive element  27  effects spectral analysis of the fluorescent signal. 
   Each of the SPD&#39;s produces at its output a stochastic stream of electrical pulses of similar shape, and information about the intensity of the received fluorescent radiation is contained in the temporal characteristics of the stochastic stream. With reference to  FIG. 2 , the temporal characteristics  210  from each SPD are registered by a recorder  211  whose structure is described below in further detail in connection with  FIGS. 4 and 5 . In a preferred embodiment, also described below in further detail in connection with  FIGS. 4 and 5 , the description of the stochastic stream of pulses is specified in terms of the time separations between arrivals of successive pulses. The detection system further comprises a signal processor unit  212  and means for transferring the recorded information at an appropriate rate from the recorder  211  to the signal processor unit  212 . 
     FIG. 2  illustrates combination of a modulated light source for excitation of fluorescence with a dispersive element for analyzing the fluorescent response into distinct spectral bands, and single-photon detection of modulated fluorescence in each of the spectral bands. Additionally, as in  FIG. 1 , the modulated light source can be multi-band also, so that the radiation flux  22  comprises a plurality of spectral bands, each modulated according to its own distinct time domain function. In this case, a preferred technique is advantageous further in that different fluorescent species are distinguished both by their fluorescence emission spectrum and their fluorescence excitation spectrum. This enhances the fidelity of fluorophore identification. 
   A preferred third embodiment of the invention, illustrated by  FIG. 3 , can be viewed as an improvement over a known technique for multicolor fluorescent detection, e.g. as applied according to the above-referenced patent to Smith et al. The known technique is combined with single-photon detection, using a modulation technique disclosed in U.S. patent application Ser. No. 08/946,414, filed Oct. 7, 1997 by Gorfinkel et al. In accordance with the latter technique, radiation reflected, transmitted, or fluorescently emitted by an object is encoded in such a way that the encoded radiation carries information about properties of the object, e.g. its color as characterized by reflected wavelengths, or the identity and quantitative content of fluorescent species present in the object. In the present embodiment of the invention, temporal encoding of different spectral components of a fluorescent signal is combined with single-photon detection of the encoded spectral components, for enhanced sensitivity. 
     FIG. 3  shows radiation  32  from optical source  31  being focused by an objective  33  onto a fluorescent sample  34 . In contrast to the embodiments illustrated by  FIGS. 1 and 2 , the optical source  31  need not be modulated, and the radiation  32  may or may not be encoded. Fluorescence  35  emitted by the sample  34  in response to incident radiation  32  is directed by an objective  36  onto an optical processor which comprises a dispersive element  37 , e.g. a prism or a diffraction grating, and a set of optical modulators  38 . The dispersive element  37  effects spectral analysis of the fluorescence  35 . The spectral components are directed onto a set of optical modulators  38  which modulate in time the resolved spectral components in such a way that each different resolved spectral component is coded by a distinct function of time. The modulated components  39  of the fluorescent spectrum are combined by an optical element  310  into an optical flux  311  focused onto the optical input of the single-photon detector  312 . The output of the detector  312  represents a stochastic stream  313  of electrical pulses of similar shape, whose temporal characteristics are registered by the recorder  314  which is described below in further in connection with  FIGS. 4 and 5 . In a preferred embodiment, also described below in further detail, the description of the stochastic stream of pulses is specified in terms of the temporal separation between arrivals of successive pulses. The detection system further comprises means  315  for transferring the recorded information at an appropriate rate to a signal processor unit  316 . 
   B. Single Photon Detection of Modulated Photon Fluxes 
   A preferred fourth embodiment of the invention is illustrated by  FIG. 4 , of a method for registration of temporal parameters of a stochastic sequence of pulses of constant or similar shape. 
   The recorder of  FIG. 4  operates with a controlled time resolution, controlled by a clock  45  which provides a regular sequence  46  of electrical pulses of constant shape which define the recording time intervals. A stochastic stream  41  of electric input pulses may originate from a sensing element of a single-photon detector which is typically a photo-multiplying tube (PMT) or an avalanche photo diode (APD). 
   The input pulses are not required to be of the same shape. With an APD, a special avalanche quenching circuit is used, either passive or active. Typically, the APD is pre-biased into its avalanche regime, for the first photon to initiate the avalanche. To prepare for the next photon arrival, the avalanche has to be quenched. It may be advantageous to use a so-called forced-quenching circuit which regularly quenches the avalanche condition, irrespective of whether an avalanche had actually been initiated, so that the arrival of photons and the time of quenching are not correlated. As a result, the avalanche-pulse duration will be stochastic also, depending on the time of photon arrival relative to subsequent quenching. 
   The stream of pulses  41  is directed to an n-state cyclic state-shift device or register  42 . Such a device has n successive stable states which may be numbered  0 ,  1 ,  2 , . . . , n- 1 , with a change from a state k to its successor state k+1 being triggered by an input pulse, and with state n- 1  having state  0  as its successor state. Between input pulses, the n-state cyclic state-shift device  42  retains its state. For example, for a 2-state cyclic state-shift device a flip-flop can be used, having a sequence of stable states  0 ,  1 ,  0 ,  1 , . . . , with each input pulse causing a transition from  0  to  1  or from  1  to  0 . It is not necessary that the cyclic state-shift device return to its initial state when its state is read. This is in contrast to conventional photon counters where each reading of the counter data is accompanied by resetting the state of the counter back to the initial state. 
   For the sake of specificity, without limiting the invention, a flip-flop will be assumed in the following further description of FIG.  4 . The output from the flip-flop represents a stochastic sequence  43  of rectangular pulses of variable length. The sequence  43  is directed to a recording device  44 , which can be realized 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  FIG. 4  operates essentially in an asynchronous mode. But, in contrast to asynchronous photon counters which record the total number of photons arriving in a particular time interval, the preferred recorder records their times of arrival. Accuracy of recording of the arrival time is controlled by the clock  45 . 
   Time intervals are recorded without measuring the duration of the intervals. This function can be performed by one of a number of devices known to those skilled in the art, placed in an electrical circuit serially with the recorder and using its output signal  47 . For example, a general-purpose computer can be used to process the array of data acquired by the recording device  44 . 
   In some applications it may be advantageous to integrate in a single device the functions of registering the time intervals between successive single photon detections and measurement of the time intervals. Such an integrated preferred fifth embodiment of the invention is illustrated by  FIG. 5 , for a stochastic stream of electric pulses  51  to which the shape and APD-quenching considerations concerning pulses  41  of  FIG. 4  are applicable also. 
   As shown in  FIG. 5 , a stochastic stream of electric pulses  51  is directed onto a flip-flop  52 . Its output represents a stochastic sequence  53  of rectangular input pulses of variable length. The sequence  53  is split three ways between counters  56  and  56 ′ and the controlled delay line  531 . The counter  56  receives the signal from the flip-flop directly, and the counter  56 ′ receives its signal through an inverter  521 . Thus, the counters  56  and  56 ′ are controlled by opposite-phase signals. Instead of a flip-flop,  52 , an n-state cyclic state-shift device can be used, as described with reference to FIG.  4 . Advantageously in this case, instead of two counters,  56  and  56 ′, up to n counters can be used. 
   A clock  54  provides a regular sequence  55  of electric pulses of constant shape which are counted by the counter  56 . Exemplarily, counter  56  is that counter whose input signal equals 1 at the time of clock pulse arrival. Advantageously, if the pulses  51  originate from and APD, the external quenching circuit which periodically forces the APD out of its avalanche regime can be synchronized by the clock  54 . There is no advantage in increasing the quenching frequency beyond the clock frequency which provides the basic discretization of time in the technique. 
   When a photon is detected and an electric pulse  51  enters the flip-flop  52 , one of the counters  56  and  56 ′ stops counting and the other begins counting. The one counter that has just stopped counting then contains the record  57  of how long the interval between two successive pulses has lasted, measured in terms of the number of clock cycles counted. The record  57  is transferred to the recording device  510  through a commutator  58  which serves to provide successive recording at intervals of time so that, while one time interval is being recorded, the next one is being measured. The commutator  58  is controlled by a switch signal which is derived by input signals  53  delayed by a characteristic time τ 1  corresponding to the response time of the counter  56 . The output of the commutator  58  represents a sequence of codes  59  describing the measured time intervals between detected photons. The codes  59  appear at the output of the commutator  58  in stochastic fashion corresponding to the detection of incoming photons and delayed by the time interval which is the sum of τ 1  and the response time τ 2  of the commutator itself. It is advantageous, therefore, to control the recording device  510  by switch signals which are derived from the input signals  53 , delayed from the moment of flip-flop switching by the time τ 1 +τ 2 . The output  514  of the recording device  510  represents the same sequence  59  of codes describing the measured time intervals between detected photons. In contrast to the sequence  59 , which is accumulated in time stochastically, the sequence  514  can be transmitted in a regular fashion, e.g. at a constant rate, for further processing. 
   Further to the technique illustrated by  FIG. 4 ,  FIG. 6  illustrates inclusion of D-triggers for minimizing the number of pulses uncounted due their close spacing in time. Electric pulses from 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 an 8-bit word or byte. To avoid changing the state of the counter  62  during storage, the synchronous pulse generator  65  shuts off the switch  61  simultaneously with sending a short record pulse to the input Wr of the storage register  63 . The output from the storage register  63  goes through the buffer  64  directly to the parallel port of a computer. Operational control error indicator is facilitate by a logic comparator  66  equipped with an LED (light emitting diode)  67 . The parallel computer port is synchronized by a synchronous pulse through a delay line  68  with a suitable delay τ. The same delayed pulse synchronizes the logic comparator  66 . 
   For an exemplary embodiment of the the technique illustrated by  FIG. 6 , the following may be specified and realized: a discretization frequency of 125 KHz, a maximum number of pulses per discretization interval of 256, a minimum time between registered pulses of 20 ns, a maximum average frequency of registered pulses of 32 MHz, and a maximum fraction of missed photons of 0.25%. 
   Techniques of the invention can be used to advantage in a variety of applications involving encoded electromagnetic radiation, including multicolor luminescent detection based on fluorescence spectroscopy and fluorescence excitation spectroscopy. They can be used in general sensor applications with other modulated luminescence signals, e.g., those based on various spectroscopic techniques such as transmission, absorption, reflection, or Raman spectra, as well as electro-luminescence, chemiluminescence and the like. The techniques are especially useful for detecting weak signals, e.g. those prevalent in optical communication links where signals are transmitted over long optical fibers.