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WO2001009605A1 - Suppression du bruit de fond en fluorometrie - Google Patents

Suppression du bruit de fond en fluorometrie Download PDF

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Publication number
WO2001009605A1
WO2001009605A1 PCT/US2000/020678 US0020678W WO0109605A1 WO 2001009605 A1 WO2001009605 A1 WO 2001009605A1 US 0020678 W US0020678 W US 0020678W WO 0109605 A1 WO0109605 A1 WO 0109605A1
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decay
molecule
sensing
time
sample
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Inventor
Joseph R. Lakowicz
Ignacy Gryczynski
Zygmunt Gryczynski
Michael L. Johnson
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

  • the present invention relates to a direct method for real-time suppression of autofluorescence in time- domain or frequency-domain fluorometry.
  • the method uses a gated detector and the sample is excited by a pulsed train.
  • the detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence.
  • Fluorescent detection is used throughout the biosciences for numerous applications including clinical chemistry, DNA sequencing, FISH, flow cytometry, high throughput screening and cellular imaging [1-8], In many instances the sensitivity is limited by interfering autofluorescence from the sample rather than detectability of the emission.
  • This interference can be suppressed with gated detection [9-10] in which the detector is gated off during an excitation pulse, and gated on following a suitable time delay which allows the scattered light and shortlived autofluorescence to decay.
  • This method is frequently used with the long lifetime lanthanides in the so-called "time- resolved immunoassays" [11] .
  • time- resolved immunoassays [11] .
  • methods to directly suppress or off-gate the short-lived interference are not available when using the frequency-domain method.
  • MLCs metal-ligand complexes
  • the present invention provides a method for frequency- domain measurement of the time-resolved intensity decays of long-lived luminophores with real-time suppression of the shortlived interfering autofluorescence.
  • the present method allows recovery of the lifetimes and amplitudes, and is not just a measurement of the integrated intensity of the long lifetime emission which is the basis of the so-called "time-resolved immunoassays" [11] .
  • the intensity decay parameters are of interest because of the possibility of chemical sensing based on the decay times [16-18].
  • the method depends on the use of a train of excitation pulses, rather than a sine wave modulated light source. It is known that a pulse train can be used for excitation in frequency-domain fluorometry based on the harmonic content of the pulses [21-24] . As an example, a 1 MHz pulse train with a pulse width near 0.3 ns has useful harmonic content at each integer multiple of 1 MHz up to about 1 GHz [22] .
  • the use of pulse train excitation provides the opportunity to turn off the detector during and immediately after the excitation pulse, at which time the emission contains the shortlived autofluorescence. This opportunity does not seem to be available with sine wave excitation. Methods to suppress single decay time components have been described [25-27], but these methods cannot be used to suppress autofluorescence and still recover the time-resolved parameters.
  • the use of short lifetime reference fluorophores was described previously as a method to correct for the wavelength-dependent time response of photomultiplier tubes (PMTs) [28-29] . If the decay time of the reference is known, the measured phase and modulation of the sample can be corrected to that which would have been observed with scattered light or with a zero decay time reference. However, such ns lifetime standards can not be used with the present background suppression method because their emission will not be detectable at longer times.
  • the present invention utilizes a reference with an adequately long lifetime so that its emission is observable until the PMT is gated on. The sample and reference are observed with the same gated PMT, with the same gating time profile. The PMT is gated on after each excitation pulse, following a delay time suitable for decay of the autofluorescence. We show by simulations that the phase and modulation data can be used directly to recover the intensity decay parameters of the long lived luminescence.
  • the present invention relates to a method for determining the presence or concentration of an analyte, comprising the steps of: a) providing a fluorescent reference molecule and a fluorescent sensing molecule; b) exposing said sensing molecule to an analyte to form a mixture, wherein said analyte is capable of changing an aspect of the fluorescence emitted by the sensing molecule in a concentration-dependent manner; c) exposing said reference molecule and said mixture to a pulsed radiation source which causes said reference and sensing molecules to emit fluorescence; d) measuring an aspect of the fluorescence emitted by said reference and sensing molecules following a delay time long enough to allow the decay of any autofluorescence from said mixture; and e) correlating the step (d) measurement with the presence or concentration of said analyte in said mixture.
  • Figure 2 depicts the fractional intensity of the background (B) and sample (S) for various on-gating times (t 0N ) .
  • the intensity decay law is given by Equation 1.
  • Figures 3 (A) -3(D) show the distortion of the frequency response by increased background.
  • the goal of gated detection is to recover the background-free intensity decay (dashed line) .
  • Figures 4 (A) -4(C) show the effect of gated detection on the emission resulting from pulse train excitation. Top, no gated detection; middle, the gating function; bottom, signal seen by the detection with gating.
  • the solid lines represent the sample signal and the dashed lines the reference signal.
  • Figures 5 (A) -5(D) show the simulated frequency-domain phase and modulation data with gated detection.
  • the parameter values are the same as on Figure 3(D) except that ⁇ s was varied from 500 to 5000 ns . From top to bottom the lifetimes recovered from the least-squares analysis were 492, 996, 1996 and 5022 ns .
  • Figures 7 (A) -7(C) show the effect of incomplete suppression of the background signal due to a large amplitude of ⁇ B .
  • the assumed on-off ratio was 7 x 10 4 .
  • Figures 8 (A) -8(D) show the effect of incomplete background suppression because of an early gating-on time.
  • B ° 1000
  • ⁇ B 10 ns
  • the gating ratio was 7 x 10 4 .
  • Figure 9 shows the dependence of observed fluorescence intensity on the on-gating time t 0N .
  • B refers to a background fluorescence, S to sample, and T to total (B+S) fluorescence.
  • Figures 10 (A) -10(B) show the measurements of multi- exponential intensity decay with gated detection.
  • the solid lines are the sample signal and the dashed lines the reference signals.
  • Figure 11A shows simulated data for a double exponential decay with ( ) and without background
  • Figure 11B shows the analysis of simulated data for a double-exponential decay with background and with gated detection. The decay, background and gating parameters are the same as on Figure 10.
  • Figure 12 shows the dependence of short component amplitude
  • Figures 13 (A) -13(D) show time-domain anisotropy data.
  • the top panel is the pulses and the second panel is the gating function.
  • the third panel shows the data with insufficient gating when the on time is at 20 ns .
  • the lowest panel shows that the correct anisotropy decay for the long-lived sample is obtained with a gating time of 40 ns, which is adequate to eliminate the background fluorescence.
  • Figures 14 (A) -14(F) show frequency-domain anisotropy data for the sample with various gating times.
  • the solid line shows the values expected for the sample without background fluorescence.
  • the top panels (A and B) show that the data are seriously distorted by the presence of a background component with a 10 ns correlation time. The data are shifted towards the correct values for the 20 ns on-time. With the 40 ns on-time the data overlapped precisely with the expected values.
  • the present invention provides a way to decrease the background or autofluorescence given off by a sample in the course of assaying for the presence or concentration of an analyte of interest.
  • the present invention may be used to detect any analyte for which a suitable fluorescent sensing probe is available, i.e., a sensing molecule whose fluorescent emission changes upon exposure to the analyte in a concentration-dependent manner.
  • a suitable fluorescent sensing probe i.e., a sensing molecule whose fluorescent emission changes upon exposure to the analyte in a concentration-dependent manner.
  • Many such molecules are well-known, and may be used to measure, for example, pH; saccharides such as glucose, fructose and other cis-diols; oxygen; blood gases; various electrolytes such as zinc, potassium, carbonate, sodium, magnesium, etc . ; proteins; nucleic acids; tissue fluorescence, etc.
  • the fluorescent reference molecule used as a lifetime reference in the present invention is chosen to have a sufficiently long fluorescent lifetime so as to allow the decay of any autofluorescence from the mixture of the sample and the sensing molecule before the fluorescence of the reference molecule is analyzed.
  • the lifetime of the reference molecule should generally be longer than the lifetime of the sample autofluorescence.
  • the lifetime of the reference molecule is at least about 20 ns, more preferably on the order of about 1 ⁇ s to about 1 ms .
  • suitable reference molecules [12-20] for example those sold by Fluka and other companies. These include transition etal- ligand complexes and lanthanide metal-ligand complexes.
  • the present invention contemplates the use of shorter lifetime reference molecules, on the order of about 2-10 ns, under certain circumstances.
  • short lifetime reference molecules would be useful when the present invention is used to analyze tissue, as there would be a great deal of scattering off of the tissue.
  • the emission from even a nanosecond lifetime reference molecule could be observable at the longer times used herein.
  • the reference molecule may be a distinct entity but having the same structure as the sensing molecule, provided that the reference molecule is not exposed to the analyte, e.g., is isolated in a separate compartment, is embedded in a matrix, or the like.
  • the sensing molecule is exposed to a sample which may contain an analyte of interest.
  • the reference and sensing molecules are then exposed to a pulsed radiation source which causes the molecules to emit fluorescence.
  • the choice of the radiation source will depend on a number of factors, such as the fluorescent characteristics of the reference and sensing molecules, the specific application, etc .
  • Preferred sources include lasers, laser diodes, light emitting diodes, arc lamps, flash lamps, electroluminescent devices, sunlight and other light sources.
  • the fluorescent emission from the reference and sensing molecules is then analyzed.
  • the time delay may be accomplished simply by conventional gated detection.
  • the sensing molecule of the present invention will generally have a detectable quality that changes in a concentration-dependent manner when the macromolecule is exposed to the analyte to be measured. Many such qualities are known and may be used in the present invention.
  • the sensing molecule may include a luminescent (fluorescent or phosphorescent) label, an absorbance based label, etc.
  • the sensing molecule may comprise an energy donor moiety and an energy acceptor moiety, each spaced such that there is a detectable change when the sensing molecule is exposed to the analyte.
  • the detectable quality is a detectable spectral change.
  • a detectable spectral change includes changes in fluorescent decay time (determined by time domain or frequency domain measurement) , fluorescent intensity, fluorescent anisotropy or polarization; a spectral shift of the emission spectrum; a change in time- resolved anisotropy decay (determined by time domain or frequency domain measurement) , etc.
  • various detection techniques also are known in the art that can be used.
  • the present invention is applicable to fluorescence lifetime imaging microscopy. In that case, an imaging detector such as an image intensifier and a CCD camera would be gated in a way analogous to a photomultiplier tube.
  • the present invention may be utilized in any environment in which analytes may be measured.
  • the present methods may utilize, e.g., nucleic acid arrays, multi-well plates, etc.
  • gated detection can alter the apparent values of lf particularly when the sample (S) decay is a multi-exponential.
  • the intensity decay of the sample displays two decay times ( ⁇ B and ⁇ s ) associated with the emission from the background (B) and from the sample (S) . The intensity decay is thus given by
  • the goal of gated detection to measure the sample decay time ⁇ s without interference from the background component is the goal of gated detection to measure the sample decay time ⁇ s without interference from the background component .
  • I obs (t) 1000 exp (-t/10 ns) + 10 exp (-t/1000 ns) (3 )
  • the experimental goal is to measure the longer decay time without interference from the short-lived autofluorescence .
  • the simulated intensity decay shows that a component of interest, with a total intensity of 50% of the measure signal, will have a minor amplitude in the time dependent decay ( Figure 1) . Stated conversely, the time-zero amplitude of the background signal can be many-fold greater than the amplitude of the component of interest.
  • ⁇ B ° the frequency responses of the background signal
  • the frequency responses ( ) become distorted from the response expected from the sample itself (— — —) .
  • the amplitude of the background can exceed that of the sample, making the signal of interest a minor component in the measured signal.
  • the fraction of the signal due to the component of interest contains more Poisson noise due to the higher intensity signal. For these reasons, it is preferable to eliminate the background signal prior to detection.
  • the gated concept can be applied to frequency-domain fluorometry.
  • the light source is a continuous pulse train ( Figure 4).
  • a pulse train is known to be useful for frequency-domain measurements when using the harmonic content method [21-24].
  • the frequency-domain data can be measured at every integer multiple of the pulse repetition frequency up to a frequency near (t p ) _1 , where t p is the pulse width of the incident light.
  • the top panel of Figure 4 shows the signal expected without gated detection.
  • the vertical dotted lines ( • • • • ) show the signal observed from the usual dilute scattering reference which displays a zero lifetime.
  • the solid lines ( ) show the intensity decay of the sample.
  • the sample intensity decay is assumed to display a short-lived signal due to background, as well as a long decay time due to the sample.
  • the short-lived component is the vertical region of the decay, and the decay of the sample of interest is the angled region of this line.
  • the reference did not display a short lived component, but this assumption is not necessary because gated detection is performed on both the sample and the reference .
  • phase and modulation When measuring the phase and modulation the sample ( ) and reference (— — —) signals are alternatively observed using the same detector.
  • the phase and modulation are typically measured using cross correlation electronics at the desired measurement frequency ( ⁇ in radians/sec) and one obtains the same phase and modulation of the emission as if the excitation source was modulated as a pure sine wave at frequency ⁇ [21-24] . Because the detector is continuously on, one observes the total emission from the short and long lifetime components, and the observed phase ( ⁇ ⁇ obs ) and modulation (m ⁇ obs ) are distorted by the presence of the short-lived background.
  • the gating function would be a sequence of rectangular gating pulses ( Figure 4, middle panel) .
  • the short-lived components do not contribute to the signal seen by the PMT.
  • the gating function will completely suppress the signal from the scattering reference, so one cannot measure the phase difference and modulation of the sample as compared to the scattering reference. While in principle one could determine in the arrival time of the pulses by other means, much of the precision and freedom from artifacts in frequency-domain measurements originates by comparing the scattering reference and the sample with the same detector under the same experimental conditions .
  • the difficulty caused by suppression of the reference signal by the gating function can be overcome using long lifetime reference luminophores .
  • the scattering solution is replaced by a reference which displays a known single exponential lifetime ( ⁇ R ) .
  • the phase and modulation of the reference, relative to a scattering reference, is given by
  • phase angle ( ⁇ obs ) for the sample is shorter than the true value by ⁇ R [28- 29] .
  • the actual phase angle ( ⁇ ) of the sample is given by
  • correction of the measured phase and modulation values for a reference lifetime is a standard part of most frequency-domain data analysis programs. References with known lifetimes are used to correct for the color-dependent time response of PMTs . Most fluorophores used as lifetime references have decay times of 1 to 10 ns [28-29] . Hence, except as noted above, these standards cannot be used with this gating method because their emission will have decayed prior to on-gating of the detector. This difficulty can be solved by using longer decay time references luminophores . In particular, the transition metal-ligand complexes display usefully long decay times near 1 ⁇ s, and frequently display single exponential decays in fluid solvents.
  • Frequency-domain measurements can thus be performed with suppression of autofluorescence by using off-gating at times near the excitation pulse.
  • the long lifetime can be obtained using eqs . 8 and 9.
  • the resulting data can be directly used in currently available frequency-domain software to recover the multiple decay times without any modification. Minor changes in the software are needed to recover the true time-zero amplitudes .
  • I B (t) describes the intensity decay of the background autofluorescence
  • I s (t) the decay law of the sample in the absence of autofluorescence.
  • the recovered decay time is the long decay time of the sample, and the amplitude assumed equal to 1.0.
  • the situation is slightly more complex for a multi-exponential decay.
  • the amplitude and frequency of the oscillation depended on the ⁇ R , ⁇ s , t 0N , and t 0FF . In practice, this does not seem to be a serious problem.
  • the autofluorescence can also distort the frequency-domain data if the gate-on time is too short compared to the decay time of the background ( Figure 8) .
  • the autofluorescence decay time is 10 ns .
  • the on-time is delayed to 150 ns the background signal is not seen in the frequency response.
  • the on-time is shortened to 100 or 90 ns, the presence of a short lived component is visually evident.
  • the on-time can be adjusted to longer times. It is straightforward to calculate the effect of the gating-on time on an assumed intensity decay.
  • Figure 13 shows time-domain anisotropy data.
  • the top panel is the pulses and the second panel shows the gating function.
  • the third panel shows the data with insufficient gating when the on time is at 20 ns . That is somewhat too short for the assumed background of the interfering signal which had a 10 ns lifetime.
  • the fourth panel shows that the correct anisotropy decay for the long-lived sample is obtained with a gating time of 40 ns, which is adequate to eliminate the background fluorescence. The parameters were as described above .
  • Frequency-domain background suppression is needed in a variety of analytical and clinical applications of time-resolved fluorescence. For instance, phase-modulation measurements with long lived metal-ligand complexes are being developed for use in resonance energy transfer immunoassays, measurements of blood electrolytes and gases, bioprocess monitoring and in high throughput screening for drug discovery. In all these applications it would be valuable to make use of the high sensitivity of gated detection with the robustness of phase- modulation fluorometry. The present invention is useful in these important applications. Table I. Effect of Off-Gating on the Fractional Intensities of the Background (B) and Sample (S)*

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Abstract

Dans l'un de ses aspects, cette invention se rapporte à un procédé direct servant à la suppression en temps réel de l'autofluorescence lors d'opérations de fluorométrie dans le domaine temporel ou dans le domaine fréquentiel. Ce procédé utilise un détecteur à déclenchement périodique et l'échantillon est excité par un train d'impulsions. Le détecteur est déclenché périodiquement à la suite de chaque impulsion d'excitation, après un délai approprié, en vue d'obtenir la décroissance de l'autofluorescence instantanée.
PCT/US2000/020678 1999-07-30 2000-07-31 Suppression du bruit de fond en fluorometrie Ceased WO2001009605A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002090948A1 (fr) * 2001-05-03 2002-11-14 Delta Dansk Elektronik, Lys & Akustik Appareil et detecteurs pouvant mesurer la duree de fluorescence utile d'un detecteur de fluorescence
WO2006089342A1 (fr) * 2004-10-18 2006-08-31 Macquarie University Detection par fluorescence
WO2007040459A1 (fr) * 2005-10-06 2007-04-12 Nanyang Technological University Elimination d'un bruit de fond en fluorescence
FR2944104A1 (fr) * 2009-08-25 2010-10-08 Commissariat Energie Atomique Extinction de l'autofluorescence des tissus biologiques en tomographie resolue en temps
US7977650B2 (en) 2006-08-02 2011-07-12 Commissariat A L'energie Atomique Method and device for 3D reconstruction of the distribution of fluorescent elements
US8193518B2 (en) 2009-09-24 2012-06-05 Commissariat à l'énergie atomique et aux énergies alternatives Device and method for spatial reconstructing of fluorescence mapping
WO2014137992A1 (fr) * 2013-03-04 2014-09-12 Rosenthal Scott Bruce Procédés et système pour mesurer une durée de vie de luminescence
US8847175B2 (en) 2010-12-15 2014-09-30 Commissariat A L'energie Atomique Et Aux Energies Alternatives Method for locating an optical marker in a diffusing medium
US9036970B2 (en) 2009-10-08 2015-05-19 Commissariat A L'energie Atomique Et Aux Energies Alternatives Method and device for diffuse excitation in imaging

Citations (4)

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US5212099A (en) * 1991-01-18 1993-05-18 Eastman Kodak Company Method and apparatus for optically measuring concentration of an analyte
US5618732A (en) * 1992-07-31 1997-04-08 Behringwerke Ag Method of calibration with photoactivatable chemiluminescent matrices
US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
US5863401A (en) * 1994-04-14 1999-01-26 Beckman Instruments, Inc. Simultaneous analysis of analytes by immunoassay using capillary electophoresis with laser induced fluorescence

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5212099A (en) * 1991-01-18 1993-05-18 Eastman Kodak Company Method and apparatus for optically measuring concentration of an analyte
US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
US5618732A (en) * 1992-07-31 1997-04-08 Behringwerke Ag Method of calibration with photoactivatable chemiluminescent matrices
US5863401A (en) * 1994-04-14 1999-01-26 Beckman Instruments, Inc. Simultaneous analysis of analytes by immunoassay using capillary electophoresis with laser induced fluorescence

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002090948A1 (fr) * 2001-05-03 2002-11-14 Delta Dansk Elektronik, Lys & Akustik Appareil et detecteurs pouvant mesurer la duree de fluorescence utile d'un detecteur de fluorescence
WO2006089342A1 (fr) * 2004-10-18 2006-08-31 Macquarie University Detection par fluorescence
US7812324B2 (en) 2004-10-18 2010-10-12 Macquarie University Fluorescence detection
WO2007040459A1 (fr) * 2005-10-06 2007-04-12 Nanyang Technological University Elimination d'un bruit de fond en fluorescence
US8173973B2 (en) 2005-10-06 2012-05-08 Nanyang Technological University Eliminating fluorescence background noise
US7977650B2 (en) 2006-08-02 2011-07-12 Commissariat A L'energie Atomique Method and device for 3D reconstruction of the distribution of fluorescent elements
FR2944104A1 (fr) * 2009-08-25 2010-10-08 Commissariat Energie Atomique Extinction de l'autofluorescence des tissus biologiques en tomographie resolue en temps
US8193518B2 (en) 2009-09-24 2012-06-05 Commissariat à l'énergie atomique et aux énergies alternatives Device and method for spatial reconstructing of fluorescence mapping
US8253116B1 (en) 2009-09-24 2012-08-28 Commissariat à l'énergie atomique et aux énergies alternatives Device and method for spatial reconstructing of absorbers mapping
US9036970B2 (en) 2009-10-08 2015-05-19 Commissariat A L'energie Atomique Et Aux Energies Alternatives Method and device for diffuse excitation in imaging
US8847175B2 (en) 2010-12-15 2014-09-30 Commissariat A L'energie Atomique Et Aux Energies Alternatives Method for locating an optical marker in a diffusing medium
WO2014137992A1 (fr) * 2013-03-04 2014-09-12 Rosenthal Scott Bruce Procédés et système pour mesurer une durée de vie de luminescence

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