United States Patent |
6,934,030
|
Luryi
,   et al.
|
August 23, 2005
|
Method and apparatus for detecting radiation
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.
Inventors:
|
Luryi; Serge (Old Field, NY);
Gorfinkel; Vera (Stony Brook, NY);
Gouzman; Mikhail (Lake Grove, NY)
|
Assignee:
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The Research Foundation of State University of New York (Stony Brook, NY)
|
Appl. No.:
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322294 |
Filed:
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December 18, 2002 |
Current U.S. Class: |
356/432; 356/436; 250/459.1; 378/44 |
Intern'l Class: |
G01N 021/00 |
Field of Search: |
356/311,432,436,317,318
378/44-49,51
|
References Cited [Referenced By]
U.S. Patent Documents
5171534 | Dec., 1992 | Smith et al.
| |
5433197 | Jul., 1995 | Stark.
| |
5565982 | Oct., 1996 | Lee et al.
| |
5784157 | Jul., 1998 | Gorfinkel et al.
| |
5793049 | Aug., 1998 | Ballard.
| |
5818057 | Oct., 1998 | Buck.
| |
5940545 | Aug., 1999 | Kash et al.
| |
6043506 | Mar., 2000 | Heffelfinger et al.
| |
6071748 | Jun., 2000 | Modlin et al.
| |
6137584 | Oct., 2000 | Seidel et al.
| |
6528801 | Mar., 2003 | Luryi et al.
| |
6760109 | Jul., 2004 | Luryi et al.
| |
Foreign Patent Documents |
9823941 | Apr., 1998 | WO.
| |
Other References
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, pp. 690-696 (1996).
W.R. McCluney, "Introduction to Radiometry and Photometry," Artech House, Jun.
30, 1994, pp. 114-122.
Alan Smith, "Selected Papers on Photon Counting Detectors," SPIE (Milestone Series),
vol. MS413, Feb. 4, 1998 (ISBN: 0-8194-2788-8), pp. 194-202, published by the Society
of Photo-optical Instrumentation Engineers (SPIE).
|
Primary Examiner: Lauchman; Layla
Attorney, Agent or Firm: Baker Botts LLP
Claims
1. A method for analyzing a sample by the detection of interacting X-rays corresponding
to radiation from the sample, comprising the steps of:
(a) detecting successive quanta of intensity modulated interacting X-rays corresponding
to radiation from the sample, with the modulation being over time in accordance
with a preselected code;
(b) determining time intervals between instances of detection of said quanta;
(c) recording a sequence of said time intervals; and
(d) comparing the recorded sequence with said code.
2. The method according to claim 1, wherein the interacting X-rays comprise transmitted X-rays.
3. The method according to claim 1, wherein the interacting X-rays comprise comprises
absorbed X-rays.
4. The method according to claim 1, wherein the interacting X-rays comprise reflected X-rays.
5. Apparatus for analyzing a sample by the detection of interacting X-rays corresponding
to radiation from the sample, comprising:.
(a) a detector moiety for detecting successive quanta of intensity modulated
interacting X-rays corresponding to radiation from the sample, with the modulation
being over time in accordance with a preselected code;
(b) a time interval determination moiety operationally coupled to said detector
moiety for determining time intervals between instances of detection of said quanta;
(c) a recorder moiety operationally coupled to said time interval determination
moiety for recording a sequence of said time intervals; and
(d) a comparator moiety operationally coupled to said recorder moiety for comparing
the recorded sequence with said code.
6. Apparatus for analyzing a sample by the detection of interacting X-rays corresponding
to radiation from the sample, comprising;
(a) detector means for detecting successive quanta of intensity modulated interacting
X-rays corresponding to radiation from the sample, with the modulation being over
time in accordance with a preselected code;
(b) time interval determination means operationally coupled to said detector
means for determining time intervals between instances of detection of said quanta;
(c) recorder means operationally coupled to said time interval determination
means for recording a sequence of said time intervals; and
(d) comparator means operationally coupled to said recorder means for comparing
the recorded sequence with said code.
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 fmod in the modulation spectrum of modulation radiation is
comparable to or higher than the average frequency fphot 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'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 τ1 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 τ1 and the
response time τ2 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 τ1+τ2. 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.
* * * * *