|United States Patent
,   et al.
July 21, 1998
Method and apparatus for identifying fluorophores
The present invention is a unique method for identifying the presence, and
preferably the identity, of a fluorophore by optically stimulating one or
more fluorophores with an optical signal which has been modulated in
intensity in the time domain. The stimulated fluorophore produces a
resulting fluorescence which is demodulated to produce an electrical
signal corresponding to the intensity modulation of the fluorescence.
Finally, the electrical signal is compared to the modulation of the
optical signal to determine whether or not the fluorophore is present. The
present method can be used alone or in conjunction with known methods of
optically analyzing fluorescence of fluorophores to determine the presence
Gorfinkel; Vera (Kassel, DE);
Luryi; Serge (Stony Brook, NY)
The Research Foundation of State University of New York (Albany, NY)
November 21, 1995|
|Current U.S. Class:
||356/318; 204/452; 204/603; 250/458.1; 250/459.1; 356/344; 356/417 |
|Field of Search:
References Cited [Referenced By]
U.S. Patent Documents
|5032714||Jul., 1991||Takahashi et al.||250/458.
|5128019||Jul., 1992||Karpf et al.
|5274225||Dec., 1993||Gorfinkel et al.
|5281825||Jan., 1994||Berndt et al.||250/458.
|5300789||Apr., 1994||Gorfinkel et al.
|5311526||May., 1994||Gorfinkel et al.
|5321253||Jun., 1994||Gorfinkel et al.
|5365326||Nov., 1994||Chrisman et al.||356/342.
|5422904||Jun., 1995||Gorfinkel et al.
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Pumping Current Control," by Gorfinkel et al., Proceedings of
International Electron Devices Meeting, (IEDM 1993 Washington, D.C.), pp.
"Rapid Modualtion of Interband Optical Properties of Quantum Wells by
Intersubband Absorption," by Gorfinkel et al., Appl. Phys. Lett., vol. 60,
pp. 3141-3143 (Jun. 1992).
"High-Frequency Modulation and Suppression of Chirp in Semiconductor
Lasers," by Gorfinkel et al., Appl. Phys. Lett., vol. 62, pp. 2923-2925
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Intersubband Absorption In Quantum Wells," by Gorfinkel et al., Quantum
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Primary Examiner: Hantis; K.
Attorney, Agent or Firm: Baker & Botts, L.L.P.
What is claimed is:
1. A method of identifying a plurality of fluorophores having a different
excitation spectra in a fluorophore containing substance comprising:
a. stimulating at least one of the plurality of fluorophores with an
incident optical signal fixed in relationship to a fluorophore substance
holder, the incident optical signal having an optical spectrum comprising
in spectral components wherein m is an integer greater than 1;
b. modulating each of said spectral components in intensity according to a
respective one of a set of linearly independent time-domain functions;
c. exciting at least one of the plurality of fluorophores with at least one
of said spectral components modulated in intensity according to said
respective one of a set of linearly independent time-domain functions to
produce fluorescence emission over a range of wavelengths;
d. providing excitation efficiencies and fluorescence quantum yields for
each of the plurality of fluorophores for each of the m spectral
e. detecting substantially all of the entire fluorescence emission excited
by the incident optical signal to produce a response signal which
corresponds to the intensity of substantially all of said fluorescence
emission over said range of wavelengths in the time-domain of the linearly
independent time-domain functions; and
f. analyzing the response signal utilizing the excitation efficiencies and
quantum yields to identify at least one fluorophore of the plurality of
2. The method of claim 1 wherein said response signal is electrical.
3. The method of claim 2 wherein a photoreceiver converting said
fluorescence emission to a corresponding electrical signal.
4. A method according to claim 1 wherein said spectral components of
incident optical signal are substantially monochromatic.
5. A method according to claim 1 wherein the wavelengths of said spectral
components of incident optical signal are known.
6. The method of claim 1 where the fluorescence emission is isolated from
the radiation corresponding to said incident optical signal.
7. A method according to claim 1 wherein said m time-domain functions are
8. A method according to claim 1 wherein said m time-domain functions are
9. The method of claim 1 wherein said response signal is in the accoustical
10. A method according to claim 1 wherein at least one of the m spectral
components of incident optical signal is modulated in intensity according
to a respective time-domain function comprising a linear combination of n
linearly independent time-domain basis functions, wherein n is an integer
greater than 1.
11. A method according to claim 10 wherein each of the m spectral
components of incident optical signal are modulated in intensity according
to a respective time-domain function comprising a linear combination of
n.sub.m linearly independent time-domain basis functions, where n.sub.m is
an integer greater than 1.
12. The method of claim 11 wherein said response signal is analyzed to
determined fluorescence lifetime.
13. A method according to claim 1 wherein each said excitation efficiency
for each fluorophore is different from the excitation efficiency of
14. A method according to claim 1 further comprising analyzing the response
signal utilizing the excitation efficiencies and quantum yields to
determine the amount of the at least one fluorophore of the plurality of
fluorophores present in the fluorophore containing substance.
15. A method according to claim 1 wherein the response signal is analyzed
where F(t) is the fluorescence excited by the incident optical signal,
.eta..sub.M is the quantum yield for a fluorophore M, .alpha..sub.KM is
the excitation efficiency of a fluorophore M excited by a spectral
component K having a wavelength .lambda..sub.K, n.sub.M is the
concentration of a fluorophore M and L.sub.K is the intensity of the
spectral component K.
16. Apparatus for detecting a plurality of flurophores in a fluorophore
containing substance comprising:
a. a source of incident optical signal having an optical spectrum
comprising m spectral components where m is an integer greater than 1, at
least one of said spectral components capable of exciting fluorophores;
b. modulator for modulating the intensity of each of said spectral
components according to a respective one of a set of linearly independent
c. a fluorophore substance holder for securing a fluorophore-containing
substance fixed in relationship to said incident optical signal and making
said fluorophore accessible to said incident optical signal from said
source and for detection of fluorescence resulting from excitation of said
d. a detector optically coupled to said fluorophore substance holder for
detection of substantially all of the fluorescence emitted by excited
fluorophores and conversion of substantially all of said emitted
fluorescence to an electrical signal; and
e. means for analyzing the electrical signal utilizing provided quantities
derived from excitation efficiencies and quantum yields for each of the
fluorophores for each of the spectral components.
17. The apparatus of claim 16 which further comprises an optical filter
element interposed between said fluorophore holder and said detector.
18. Apparatus according to claim 12 wherein said optical filter element
comprises optical receiving fiber having a refractive index profile along
the axis of the fiber with desired optical filter characteristics.
19. Apparatus according to claim 18 wherein said optical filter element
comprises a Bragg reflector.
20. Apparatus according to claim 17 wherein said optical fiber element
comprises a Fabry-Perot resonator.
21. The apparatus of claim 16 which comprises at least one transmitter
optical fiber and at least one receiver optical fiber which are fixed in
relation to said fluorophore substance holder for irradiating said
fluorophore-containing substance and for detecting said fluorescence
resulting from excitation of said fluorophore.
22. The apparatus of claim 21 where said fluorophore substance holder is a
capillary and said optical transmitter and receiver fibers are fixed in
relation to said capillary by means of T-shape connector.
23. The apparatus of claim 22 wherein said T-shape connector has degrees of
freedom for three-dimensional alignment of said optical transmitter and
receiver fibers in relation to said capillary.
24. Apparatus according to claim 16 wherein the means for analyzing the
electrical signal analyzes the electrical signal according to
where F(t) is the fluorescence excited by the incident optical signal and
converted by the detector into the electrical signal, .eta..sub.M is the
quantum yield for a fluorophore M, .alpha..sub.KM is the excitation
efficiency of a fluorophorc M excited by a spectral component K having a
wavelength .lambda..sub.K, n.sub.M is the concentration of a fluorophore M
and L.sub.K is the intensity of spectral component K.
The present invention relates to the art of information retrieval using
substances which respond to radiation, and, in particular, to
identification of the presence of a fluorophore in a medium.
BACKGROUND OF THE INVENTION
Certain substances are known to possess a unique quality of producing light
in response to being irradiating. These substances, which are referred to
herein collectively as fluorophores, produce light after being excited by
radiant energy. This property is referred to as fluorescence.
Fluorescence occurs when electrons, which have been displaced to excited
states by energy absorbed during radiation, return to lower energy levels.
Energy in the form of electromagnetic quanta is given off when the
electrons return to lower energy levels. Fluorescence begins when the
fluorophore is irradiated and ends when irradiation ceases, with a short
time delay, typically 0.1-10 ns. The intensity of fluorescence is usually
proportional to intensity of irradiation, unless the irradiation intensity
is too high.
The ability of certain substances to fluoresce has been found useful in
conducting chemical and biological analysis. In U.S. Pat. No. 5,171,534 to
Smith, et al., a system for electrophoretic analysis of DNA fragments
produced in DNA sequencing is disclosed, wherein characterization of the
fragments depends on the fluorescent property of four chromophores tagged
to the DNA fragments. The Smith, et al. technique relies on the optical
characteristics of the emission spectra of the four fluorophores used as
tags. Consequently, the Smith, et al. technique suffers from many
shortcomings associated with analysis which depends on the optical
properties of emission spectra.
For example, the Smith, et al. technique requires dyes which must have high
extinction coefficients and/or reasonably high quantum yields for
fluorescence. Apparatus required to identify the fluorophore-tagged
fragments is complex and requires accurate optical means to distinguish
the different emission spectra. Moreover, Smith, et al. is inherently
inefficient since it requires reduction of portions of the optical signal
by refining the observed emission using filtration and reducing scattered
It is, therefore, an object of the present invention to eliminate the
drawbacks of using the optical characteristics of fluorescence as a means
for conducting chemical and biological analysis.
It is another object of the present invention to conduct high speed
automated data acquisition using the fluorescence characteristics of
fluorophores with a high degree of confidence and without the need for
These and other objects will be apparent to those skilled in the art in
view of the following disclosure. Accordingly, the scope of the claimed
invention is not to be limited by the recitation set forth hereinabove.
SUMMARY OF THE INVENTION
The present invention is a new method of identifying the presence of a
fluorophore. The new method utilizes the unique characteristic of
fluorophores to emit light in response to incident radiation. According to
the present invention an optical signal having a time-domain-modulated
intensity is used to irradiate a substance which contains a fluorophore.
The fluorophore must be in an environment in which it can be freely
excited and fluoresce, and the resulting fluorescence must be detectable
by an optically sensitive receiver.
The optical signal must also be capable of exciting the fluorophore, and is
preferably a monochromatic light having a known wavelength. The
fluorophore produces a responsive fluorescence which has an intensity also
modulated in the time-domain corresponding to the modulation of the
optical signal which is used to excite the fluorophore.
The time-modulated fluorescence is then demodulated by a optically
sensitive receiver, such as a photodetector to produce a response signal,
e.g., an electrical signal or an accoustical signal, which, as a function
of time, corresponds to the intensity of the fluorescence in the
In the broadest sense, the invention contemplates using the information to
determine one or more characteristics of the fluorophore and/or its
environment. Otherwise, the resulting electrical signal can be compared to
the time-domain-modulation used to modulate the optical signal whereby the
presence of the fluorophore can be determined. In a preferred embodiment
of the identity of fluorophore can also be determined.
The optical signal used to excite the fluorophore can be modulated with an
analog characteristic or can be digitally modified.
Information regarding the fluorophore can also be obtained by
phase-resolved measurements employing multiple frequencies. Thus, a
fluorophore can be stimulated to reduce fluorescence which has "n"
distinguishable time-domain-funtions, wherein n is greater than 1, and
wherein the functions are linearly independent in time. The response
signal can be analyzed to determine the fluorescence lifetime.
The present invention also includes an apparatus for detecting fluorophores
which includes a source of optical signal capable of exciting
fluorophores, e.g., a laser, a modulator for modulating the optical signal
and connected to the source for producing the signal in order to impose
the time-domain-modulation on the signal. The apparatus also includes a
fluorophore substance holder to secure the fluorophore material and make
it available for irradiation and also for detection by a detector. A
demodulator is connected to the holder to detect the fluorescence from the
excited fluorophores and for conversion to a response signal such as an
electrical signal. Preferably the demodulator is a photoreceiver.
In one preferred embodiment of the invention, a comparator is also used in
the apparatus and is connected to the demodulator for receipt of the
signal, and can also be connected to the modulator for comparing the
modulation signal to the electrical signal in order to determine the
presence of the fluorophore.
In one preferred embodiment of the present invention, the apparatus can
include an integrated optical probe which has at least one transmitter
optical fiber and at least one receiver optical fiber and is fixed in
relationship to the fluorophore substance holder in order to introduce
incident radiation on the substance and to detect fluorescence resulting
from excitation of the fluorophores.
Furthermore, the apparatus can include an optical filter element interposed
between the fluorophore holder and the demodulator. This can be part of
the optical probe or can be separate. The optical filter element can be an
optical fiber provided with a refractive index grading profile, to
produce, e.g., either a Bragg reflector, or a Fabry-Perot etalon.
As a result of the present invention, the ability to determine the presence
of a fluorophore is significantly increased. The present technique
enhances the signal-to-noise ratio by more than tenfold, and increases the
confidence level of base pair identification in automated DNA sequencing.
Furthermore, a high-throughput miniaturized automated data acquisition
system can be provided which has a modular structure designed for use with
most fluorescence-based electrophotetic arrangements.
Consequently, high confidence level, low cost DNA sequencing systems are
achievable as a result of the present invention.
These and other advantages will be appreciated by those skilled in the art
in view of the detailed description and the drawings set forth herein. The
scope of the invention will be pointed out in the claims appended hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of the data acquisition system in accordance with the
present invention which utilizes multicolor excitation by
FIG. 2 is a schematic depicting exemplary absorption and fluorescence
spectra of two infrared dyes;
FIG. 3 is a schematic depicting the phenomenon of luminescence with
multiple monochromatic light sources;
FIG. 4 is a schematic of a universal data acquisition system in accordance
with the present invention;
FIG. 5 depicts a T-shaped fiber-capillary connector of the present
FIG. 6 depicts a fiber-receiver with a refractive index grading forming a
set of one or more Bragg reflector for rejecting the stray (scattered or
reflected) laser radiation; and
FIG. 7 depicts a fiber-receiver with a refractive index grading forming a
Fabry-Perot resonator tuned to a desired wavelength of fluorescent
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a unique method of identifying the presence of a
fluorophore which is particularly useful in conducting analysis especially
in the area of biotechnology. A fluorophore as used herein means any
moiety capable of emitting fluorescence in response to an optical signal.
The present invention also requires that the fluorophore be in an
environment in which it can be freely excited to produce its
The present invention is of particular interest in the area of automated
DNA sequencing. The development of reliable methods for sequence analysis
of DNA and RNA is key to the success of recombinant DNA and genetic
engineering technology. Previous DNA sequencing methods known to date have
relied on, among other things, the optical characteristics of fluorophores
which are used to tag DNA fragments. As previously mentioned, U.S. Pat.
No. 5,171,534 to Smith, et al. discloses irradiating DNA fragments tagged
with fluorophores to produce a characteristic fluorescence. The optical
characteristics of the fluorescence are then analyzed to determine the
presence of the fluorophore, and, consequently, information relating to
the DNA fragment being analyzed.
Many drawbacks exist with respect to the known technology, especially since
the technique involves relatively inefficient use of the fluorescence
capability of the fluorophore as well as reliance on human intervention to
In the present invention, however, detailed analysis of DNA sequencing can
be conducted completely (to include final base-pair identification)
without requirement for human intervention and with a high degree of
In accordance with the present invention an optical signal having a
modulated intensity in the time domain is used to irradiate a fluorophore
which can be excited by the selected signal. Specifically, one or more
lasers radiating at a peak wave length of the absorption spectrum of
individual labels and modulated as a distinguishable function of time,
e.g., sinusoidally at a distinct radio frequency, is used to excite the
fluorophores. Separation of the responses from one or more labels is
accomplished in the electric domain. The method used herein is
non-selective with respect to the wavelength of fluorescence.
Consequently, the present technique makes full use of each fluorescent
molecule by detecting the entire fluorescent power spectrum emitted by
each fluorophore. This is a departure from techniques used in the past,
especially the Smith, et al. technique, which rely on detection of a
narrow wave length band.
The fluorophores useful in the present invention can be selected from dyes
which are available based on criteria known to those skilled in the art.
Dyes can be selected based on the feasibility of coupling the dyes to each
of the four different dideoxynucleotides, a determination of whether or
not the modified nucleotides impacts negatively on DNA synthesis, and
whether or not any of the di-nucleotide combinations interfere with the
DNA secondary structure, and/or can be used to decrease the problems
associated with abnormal DNA migration which occurs during
In the past, a single radioactive or fluorescent label has been used to
identify all bands on the gels. This necessitates that the fragment sets
produced in the four synthesis reactions be run on separate gel tracts and
leads to problems associated with comparing band mobilities in the
different tracts. Clearly this system is inefficient and overcoming the
problems associated therewith has been the key accomplishment of the
invention by Smith et al, which ensured its wide application. The present
invention offers an alternative method for realizing multicolor labeling
which accomplishes essentially the same goal and, at the same time, offers
several technical advantages, such as higher excitation efficiency of
individual fluorophores, and better utilization of the fluorescent
radiation. Furthermore, the present detection scheme can be advantageously
combined with the known Smith et al. method, so as to enhance the signal
to noise ratio to the level where a fully automated readout system becomes
In the present invention, each tagged primer can be paired with one of the
dideoxynucleotides and used in the primed synthesis reaction.
Representative of such amino-reactive dyes include the following:
fluorscein, isothiocyanage (FITC, .lambda..sub.max.sup.Ex =495,
.lambda..sub.max.sup.Em =520, .epsilon..sub.495.perspectiveto.
8.times.10.sup.4), tetramethyl rhodamine isothiocyanate (TMRITC,
.lambda..sub.max.sup.Ex =550, .lambda..sub.max.sup.Em =578,
.epsilon..sub.550.perspectiveto. 4.times.10.sup.4), and substituted
rhodamine isothiocyanate (XRITC, .lambda.=580, .lambda..sub.max.sup.Em
=604, .epsilon..sub.580.perspectiveto. 8.times.10.sup.4) where .lambda. is
the wavelength in nanometers, Ex is excitation, Em is emission, max is
maximum, and .epsilon. is the molar extinction coefficient. These are the
dyes which were used in the Smith, et al. system. However, it is to be
clearly understood that the present invention is not limited by any
particular set of dyes and those skilled in the art will undertake to
select dyes based on criteria set forth above as well as ease of
preparation and other operational and critical criteria. Moreover, the
fluorophores are to be maintained in an environment useful to those
skilled in the art such as slab-gel, capillary, ultra-thin gel and
In any event, the present invention contemplates the effective coupling of
the fluorophore to a nucleotide in a manner which permits the nucleotide
to be contained in a medium wherein it can be freely excited and is able
to fluoresce uninhibitedly to provide the full spectra of fluorescence.
Referring to FIG. 2, the absorption or excitation spectrum and the
fluorescence spectrum of two infrared dyes identified as CY5 and CY7 are
depicted. The solid lines depict the absorption or excitation spectrum of
wavelengths while the dashed lines show the emission or fluorescence
spectrum of wavelengths.
FIG. 1 depicts a system in which the technique of the present invention can
be implemented. For purpose of explanation, consider that two lasers,
laser 1 and laser 2 are modulated in the time domain at frequencies
f.sub.1 and f.sub.2, respectively. Conventional techniques for modulation
of semiconductor lasers by varying the pump current are capable of
producing modulation frequencies up to approximately 20 GHz. Still higher
modulation frequencies can be realized by exploratory techniques,
currently under intense development, see, for example, V. B. Gorfinkel, S.
Luryi, "High-Frequency Modulation and Suppression of Chirp in
Semiconductor Lasers", Appl. Phys. Lett., 62, pp. 2923-2925, (1993); V. B.
Gorfinkel, S. Luryi, "Article that comprises a semiconductor laser, and
method of operating the article" (dual modulation), U.S. Pat. No.
5,311,526 (1994); V. B. Gorfinkel, S. Luryi, "Article Comprising Means for
Modulating the Optical Transparency of a Semiconductor Body, and Method of
Operating the Article", U.S. Pat. No. 5,300,789 (1994); V. B. Gorfinkel
and S. Luryi, "Fast data coding using modulation of interband optical
properties by intersubband absorption in quantum wells", Quantum Well
Intersubband Transition Physics and Devices, ed. by H. C. Liu, B. (1995);
and V. B. Gorfinkel, S. A. Gurevich, "Method of and means for controlling
the electromagnetic output power of electrooptic semiconductor devices",
U.S. Pat. No. 5,274,225 (1994). Also, techniques for generation of
powerful picosecond optical pulses can be found in V. B. Gorfinkel and
Serge Luryi, "Rapid modulation of interband optical properties of quantum
wells by intersubband absorption", Appl. Phys. Lett. 60, pp. 3141-3143
(1992), V. B. Gorfinkel, S. Luryi, "Article that comprises a semiconductor
laser, and method of operating the article" (dual modulation), U.S. Pat.
No. 5,311,526 (1994), V. B. Gorfinkel, S. Luryi, "Article Comprising Means
for Modulating the Optical Transparency of a Semiconductor Body, and
Method of Operating the Article", U.S. Pat. No. 5,300,789 (1994), and V.
Gorfinkel, G. Kompa, M. Novotny, S. Gurevich, G. Shtengel, I. Chebunina,
"High-frequency modulation of a QW diode laser by dual modal gain and
pumping current control." Proceedings of Int. Electronic Dev.
Meeting/IEDM'93/, 5-8 Dec., Washington, D.C., pp. 933-937; (1993). Laser 1
emits a modulated signal having a characteristic wavelength of
.lambda..sub.1 and laser 2 emits a time-modulated signal having a
characteristic wavelength of .lambda..sub.2. Infrared dyes CY5 and CY7 are
suspended in a medium which permits free excitation and luminescence. A
photodetector is situated to receive the full spectrum of fluorescence of
each of the fluorophores except the wavelengths lambda 1 and lambda 2
corresponding to the radiation of lasers 1 and 2, respectively. This is
accomplished by a rejection filter for the purpose of isolating the
photodetector from the scattered and reflected laser radiation. The full
spectra of fluorescence is depicted by the dashed lines in FIG. 2.
The photodetector demodulates the full fluorescence spectra and converts
the optimal signal to a corresponding electrical signal. The electrical
signal can then be processed by the known methods to identify the
presence, isolate, or detect the signals at frequencies f1 and f2. The
processing can be done in the electrical domain by analog techniques or
For ideal detection it would be preferable that the excitation spectra of
the two dyes were non-overlapping, so that, e.g., the radiation of laser 1
at lambda 1 would excite only fluorophore A and not fluorophore B, while
the radiation of laser 2 at lambda 2 would excite only fluorophore B and
not fluorophore A. This ideal case would completely eliminate the
"cross-talk" or parasitic excitation of a "wrong" fluorophore. The
photodetector receives the entire fluorescent radiation from both dyes A
and B. The non-fluorescent radiation from lasers 1 and 2 modulated at
frequencies f1 and f2, respectively, has been filtered out by the
rejection filter. Therefore, the presence in the detected signal of
frequency components f1 or f2 would be direct evidence of the presence of
fluorophore A or B, respectively.
However, the ideal case may be difficult to realize, because of the absence
of suitable fluorophores. Consider, therefore, the more realistic case,
when each of the lasers excites both dyes, but to a different degree.
In this case, the concentration of dyes A and B in the observation slot
varies in time as n.sub.A (t) and n.sub.B (t). The slot is illuminated by
the output L.sub.1 (t)=L.sub.f1 exp(2.pi.if.sub.1 t) and L.sub.2
(t)=L.sub.f2 exp(2.pi.if.sub.1 t), of two lasers that have wavelengths
.lambda..sub.1 and .lambda..sub.2, respectively, and are modulated at
(radio) frequencies f.sub.1 and f.sub.2.
This gives rise to the excited populations n*.sub.M (M=A,B) of the
fluorophores in the observation slot which produce fluorescence at the
rate .eta..sub.A n*.sub.A and .eta..sub.B n*.sub.B, according to the
quantum yields .eta..sub.M (M=A,B). The resultant fluorescence signal,
F(t), is of the form
where .alpha..sub.kM is the excitation efficiency of the fluorophore M by
the laser of wavelength .lambda..sub.k (k-1,2). The efficiency parameters
.alpha..sub.kM are proportional to the absorption coefficient of M at
.lambda..sub.k and the lifetime of the excited state of molecule M.
Because of mobility shifts, both A and B bands may appear in the
observation slot at the same time and be excited by both lasers to a
different degree, as given by the matrix .alpha..sub.kM.
The entire signal F(t), produced by both kinds of molecules as excited by
both lasers, is received by the photodetector. It may appear that since
the received wavelength is not optically resolved, the information
regarding the concentrations n.sub.A (t) and n.sub.B (t) is irretrievably
lost. However, we recall that the received signal contains the response to
two lasers modulated at different frequencies. Explicitly, we have:
F.sub.f.sbsb.1 =(.alpha..sub.1A .eta..sub.A n.sub.A +.alpha..sub.1B
.eta..sub.B n.sub.B)L.sub.f.sbsb.1 ; II
F.sub.f.sbsb.2 =(.alpha..sub.2A .eta..sub.A n.sub.A +.alpha..sub.2B
.eta..sub.B n.sub.B)L.sub.f.sbsb.2 ; III
Separation of these components in the electric domain is both efficient and
exceedingly accurate. It can be done with high-quality narrow-band
filters, as in a radio receiver with preset "stations". A better solution
is to use heterodyne detection, with the local oscillator signal taken
from the same source that drives the lasers in the first place.
Schematically, the frequency detection scheme with multicolor illumination
is illustrated in FIG. 1. After the signal processing, e.g., by Fourier
transform, the signal represents the amplitudes F.sub.f1 and F.sub.f2
--slowly varying functions of time, from which the concentrations n.sub.A
.tbd.n.sub.A (t) and n.sub.B .tbd.n.sub.B (t) are determined by solving
the system of equations II and III. Evidently, the scheme is generalized
to an arbitrary number of colors in a straightforward manner.
In a preferred embodiment, the present invention can include a combination
of time-intensity-modulation of fluorescence and a wavelength-selective
Cross-talk between different information channels in the conventional
wavelength-selective scheme arises due to the fact that fluorophores of a
particular kind (say, A labels) produce light not only at the wavelength
.lambda..sub.A ', referring to FIG. 3, corresponding to the fluorescence
peak of A labels, but also at other wavelengths, selected by the optical
filters at .lambda..sub.C ', .lambda..sub.T ', and .lambda..sub.G ', as
illustrated in FIG. 3. The parasitic signals are proportional to the
overlap of the fluorescence spectra of the different dyes. See FIG. 2.
Fortunately, once the fluorescence has been converted to a robust
electrical signal composed of the full spectra from all excitations,
electrical operations can be performed which discriminate the contribution
of each of the fluorophoretic emissions. A four component system will be
used to demonstrate the present technique.
In the case of four lasers (L.sub.k), the fluorophores are identified with
label k=A, C, T, or G. In other words, L.sub.A is the output radiation of
the laser "A", whose wavelength .lambda..sub.A is at the peak of the
absorption spectrum of label A. The radiation L.sub.k is modulated at the
radio frequency f.sub.k, viz. L.sub.k (t)=L.sub..lambda.,fk
Irradiation of the observation slot by the four lasers gives rise to an
excited population of the fluorophores,
where both indices k and M run over the same set of labels A, C, T, and G.
The excited molecules produce fluorescence at the rate .eta..sub.Mj
n.sub.M * where .eta..sub.Mj is the quantum yield coefficient of the
fluorophore M into the wavelength channel .lambda..sub.j '. The index j is
also labeled by the fluorophore name, j=A, C, T, or G.
The total fluorescent signal received in the channel .lambda..sub.j ' is
If we were not able to discriminate between signals induced by different
lasers, then our received signal structure would be described entirely by
Eq. (V)--which is essentially similar to the conventional four-color
detection scheme with wavelength selection. Determination of the
quantities of interest, n.sub.M (t), is accomplished by operating on the
signal to solve the system of four equations (V). The quality of data
analysis is essentially dependent on the fact that the off-diagonal
coefficients .eta..sub.Mj, (j.noteq.M) are smaller than the diagonal
coefficients .eta..sub.MM :
Preferably, the conventional detection scheme will be enhanced when the
wavelengths .lambda..sub.j ' are spread apart so as to minimize the
fluorescence overlap .phi..sub.Mj. Simply, the scheme of modulated 4-color
excitation, described above relies on the smallness of the absorption
overlap, which implies that the off-diagonal coefficients .alpha..sub.kM
(k.noteq.M) are smaller than the diagonal coefficients .alpha..sub.MM :
and the user benefits when the absorption peaks .lambda..sub.k are spread
so as to minimize .epsilon..sub.kM.
When the wavelength-selective detection of multicolor fluorescence is
combined with frequency-selective detection of modulated multicolor
excitation, then the information comes to us across 4.times.4=16 channels.
Denoting by F.sub..lambda. '.sub.j,fk (t) the amplitude of the signal
received by detector at wavelength .lambda..sub.j ' after heterodyning
with the local oscillator frequency f.sub.k the structure of the received
data can be represented in the form:
where S.sub.jk (t) are slowly varying functions of time, S.sub.jk
.tbd.F.sub..lambda. '.sub.j,fk /L.sub..lambda.k,fk. The 16 equations
(VIII) with 4 unknowns n.sub.M =n.sub.M (t) form an overdetermined system.
Needless to say, the over-determination can be used to improve the signal
to noise ratio. However, it is easy to see a dramatic improvement even if
the full overdetermined stream of information is not used--but only its 4
diagonal channels, S.sub.kk, are used. Using only diagonal elements means
that identification is performed of frequency f.sub.k only in the optical
channel, corresponding to wavelength .lambda..sub.j ' with j=k. This leads
to the four equations
Simple observation of the matrix in the right hand side of (IX)
reveals that all small parameters that were of first order in the
conventional scheme, have become of second order in smallness. The
resultant signal improvement is as if the overlaps of the fluorescent
spectra in the conventional scheme were reduced by additional small
factors .epsilon.; or as if in the multicolor modulated excitation scheme
the overlap of absorption spectra were reduced by additional small factors
.phi.. Further improvement of signal acquisition is still available by
using the "redundant" information which arrives in the 12 off-diagonal
channels--in signals that are small to first order (by either the factor
of .phi. or .epsilon.).
An advantage of the alternative combined data acquisition scheme is
illustrated schematically in FIG. 3 in the instance of the single group of
fragments, labeled "A", passing through the observation slot. Simultaneous
illumination by all four lasers results in a modulated fluorescence at all
four frequencies. One of these signals (at wavelength .lambda..sub.A ',
modulated with the frequency f.sub.A) is "strong", the rest are weaker by
the corresponding .epsilon.. Compared to the signal detected at the
wavelength .lambda..sub.A ' and heterodyned with the local oscillator
frequency f.sub.A, all other "diagonal" signals (.lambda.'.sub.M, f.sub.M,
M=C, G, or T) are weaker by the factors .epsilon..phi., which are second
order in smallness.
Compared to either the conventional scheme, e.g., Smith, et al., which
identifies labels by their fluorescent wavelength, or the invention
frequency modulation scheme of FIG. 1, which identifies the same labels by
their absorption wavelength, the combined method is somewhat more complex.
However, the combined technique is capable of suppressing the channel
cross-talk by at least one order of magnitude. Implementation of this
technique provides for the realization of a fully automated data
Phase-resolved measurements employ a (radio) frequency modulated optical
signal with a synchronous detection of the fluorescent response. The
fluorescence lifetime, .tau..sub.F, is determined from the phase shift
between the detector and the source. Difference in the .tau..sub.F can be
used for discriminating between dyes that do not possess an appreciable
difference in their fluorescence spectra F. V. Bright and L. B. McGown,
"Four Component Determinations Using Phase-Resolved Fluorescent
Spectroscopy," Anal. Chem., Vol. 57, pp. 55-59 (1984). Lakowicz and
coworkers have demonstrated that 100% contrast discrimination can be
accomplished with two dyes that differ only in their fluorescent lifetime,
.tau..sub.F =4 ns and .tau..sub.F =1.6 ns (J. R. Lakowicz and K. W.
Brendt, "Lifetime selective fluorescence imaging using an rf
phase-sensitive camera" Rev. Sci. Instrum. 62, pp. 1727-1734, (1994); and
J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Brendt and M. Johnson,
"Flurescence lifetime imaging", Nucleic Acids Res. 18, pp. 4417-4421,
(1992)). Phase-resolved detection suppresses the noise brought about by
the parasitic fluorescence from (from glass, gel, fluids, etc.) provided
the .tau..sub.F of the parasitic signal is different from that of the
Phase detection methods are ideally suited for use with semiconductor
lasers modulated at multiple frequencies. In the above discussion of
frequency-selective techniques, it has been assumed that each excitation
wavelength .lambda..sub.k generated by laser L.sub.k has a one-to-one
correspondence to the frequency f.sub.k at which this particular laser is
modulated. Of course, it is precisely this correspondence which enables us
to decode the origin of excitation. However, there is no reason, why there
could not be more than one frequency associated with a given laser and the
same wavelength. For example, communication lasers are typically modulated
at a number of "carrier" frequencies with a total bandwidth of about 550
MHz, which permits the implementation of nearly 100 parallel channels (V.
B. Gorfinkel and S. Luryi, "Fast data coding using modulation of interband
optical properties by intersubband absorption in quantum wells," Quantum
Well Intersubband Transition Physics and Devices, ed. by H. C. Liu, B.
(1995)) with exceedingly demanding specifications on the cross-talk (for
example, in a cable TV laser the combined intermodulation distortion from
all channels into a given channel cannot exceed -60 dB relative to the
carrier power in that channel).
A Multi-frequency grid method is straightforward and can be used as a
model. Schematically the set of excitation signals is listed below:
Laser Wavelength Modulation frequencies
f.sub.A.sup.(1), f.sub.A.sup.(2), . . .
f.sub.C.sup.(1), f.sub.C.sup.(2), . . .
f.sub.G.sup.(1), f.sub.G.sup.(2), . . .
f.sub.T.sup.(1), f.sub.T.sup.(2), . . .
The method is quite analogous to the well-developed technique of parameter
extraction for device equivalent circuits, widely used in electronics
(see, e.g., G. Kompa, "Modelling of dispersive microwave FET devices using
quasi-static approach," International Journ. of Microwave and
Millimeter-Wave Computer Aided Eng. 5, pp. 173-194 (1995)). With the
parallel heterodyne detection of the set of responses at multiple
frequencies we obtain not only the information about the relative
amplitudes of the excitation at different wavelengths, but also the
frequency dependence of the response to the same excitation wavelength.
This information permits determination of the phase delay with a high
accuracy. Moreover, this technique works well even with 2.pi.f.sup.k.sub.M
.tau..sub.F <<1. This feature is of particular interest in connection with
sequencing environments where the effective .tau..sub.F is quenched to
values as short as 0.1 ns--where the "conventional" phase detection would
require special current drivers and a readout circuitry operating in the
range of 1-2 GHz.
A preferred data acquisition system with both multicolor excitation and
multicolor detection is illustrated in FIG. 4. Four laser sources with
intensity-modulated output are selected so as to provide the most
efficient excitation of four fluorescent dyes. The laser radiation can be
coupled into optical fibers, which are combined in a fiber bundle which
delivers the radiation to the area of electrophoretic separation.
Depending on the power requirements, a fiber splitter can be used to split
the radiation into N channels, for parallel illumination of N sequencing
lanes. Each fiber transmitter carrying the modulated radiation is packaged
into an integrated fiber-optical probe--one probe per each lane (or
capillary) of the sequencing machine. The fiber-transmitter structure
permit focus of the radiation on a narrow spot (50-100 .mu.m). The probe
not only delivers the signal to the observation slot, but can also collect
the fluorescent response from the slot. The response signal is then
transmitted by fiber receiver to photodetectors, using the wavelength
separation via optical fibers endowed with Fabry-Perot etalons
(narrow-band pass filters) which are adapted to the fluorescent spectra of
the four dyes. Narrow-band rejection filters can also be used to cut off
spurious reflections of the laser radiation. The narrow-band pass filters
and rejection filters are described hereinbelow in connection with FIGS. 6
The photodetector demodulates the optical signal converting it to an
electrical signal. The electrical signal is processed by a special-purpose
microprocessor which provides the separation and amplification of
different frequency components and the analog-to-digital conversion. The
processed signal is delivered to a computer platform, where the automated
base calling can be performed by specially developed software tools.
The proposed system structure permits the realization of all of the
fluorescence detection methods discussed herein. The system will permit
operation in the scanning mode, although this is not the preferred mode in
view of the availability of inexpensive semiconductor lasers. The system
can also be used with non-semiconductor lasers, which is essential for an
early testing of different modules. Thus, the output of an argon-ion
laser, radiating several wavelengths simultaneously, can be split between
several channels and modulated at radio frequencies with external
The most developed class of semiconductor lasers used herein operates in
the red and near infrared spectral range (wavelength .lambda. between 0.6
mm and 1.6 mm). Due to the high power density and reasonable spatial
coherence of these lasers, it is possible to double their optical
frequency into the blue part of the spectrum with the help of nonlinear
crystals. However, the most significant potential for the use of
semiconductor lasers lies in the fact that new fluorescent dyes can be
excited in the red and near infrared. The wavelength range of these dyes
(.lambda. between 0.65 mm and 0.8 mm) can be covered by commercially
available semiconductor lasers.
Readily available red and infrared lasers work at room temperature. They
are pumped by low-voltage current sources, generating up to 100 mW of
power in the continuous wave (CW) regime. Semiconductor lasers have a
small volume (typical dimensions 300.sub..times. 100.sub..times. 100
.mu.m.sup.3). Together with a heat sink, the laser package is usually
smaller than 1 cm.sup.3. From an ultra-narrow emitting area (about 10-20
.mu.m.sup.2) they emit a very high radiation power density (up to
megawatts MW/cm.sup.2). Because of the small area of emission, the
semiconductor laser radiation can be easily focused on a small spot.
Semiconductor lasers enable a wide variety of signal manipulations, which
enhance the signal to noise ratio. Thus, one can modulate the amplitude of
semiconductor laser radiation at frequencies of 10 GHz and even higher,
generate, code, detect, and process arbitrary sequences of short pulses
with the repetition rate of up to several Gbit/s, as well as generate
ultra-short and powerful picosecond optical pulses.
It is clear that these features alone make semiconductor lasers attractive
for use in DNA sequencing systems. Moreover, semiconductor lasers open new
technical possibilities for the implementation of ultra-high performance
data acquisition. These possibilities are based on the selective
excitation of fluorescent dyes by an array of semiconductor lasers with
different radiation wavelengths and output power modulated at different
Selective excitation of fluorescence by a laser array can be accomplished
by using an array of semiconductor lasers emitting at different
wavelengths. Several different semiconductor lasers can be combined in a
compact array. Taking infrared dyes with essentially different absorption
peaks ›such as BDS dyes CY5 (.lambda.=650 nm) and CY7 (746 nm) or LI-COR
dyes IRD-40 (769 nm) and IRD-41 (787 nm)! and choosing the appropriate
radiation power ratio of the array laser, all four dye labels can
essentially be balanced. By modulating the output radiation of the array
lasers at different modulation frequencies, and using a synchronized
signal processing technique, it is possible to significantly enhance the
signal to noise ratio. Moreover, it appears feasible to detect and
electronically separate the signals from different dyes, thus dramatically
simplifying the optical part of the detection system.
Photomultiplier systems (J. A. Luckey, H. Drosman, A. J. Kostichka, D. A.
Mead, J. D'cun, T. B. Norris and L. M. Smith, "DNA sequencing analysis of
five genes ins A, B, C, D and E required for" Nucleic Acids Res., 18, pp.
900-903, (1990)) and photodiode arrays (J. C. Gluckman, D. C. Shelly and
M. V. Novotny, "Miniature fotometric photodiode array detection system for
capillary chromatography," Anal Chem, 57, 1546-1552 (1985)), intensified
photodiode arrays (D. F. Swaile and M. J. Sepaniak, J. Microcolumn
Separations, 1, 155-158 (1989)), and charge coupled device camera systems
(Y. F. Cheng, R. D. Picard and T. Vo-Dinh. "CCD CZE charged coupled device
fluorescencedetection for capillary zone electrophoresis," App. Spectrosc.
11, pp. 755-765 (1990); J. V. Sweedler, J. B. Shear, H. A. Fishman, R. N.
Zare and R. H. Scheller, "Fluorescence detection in capillary zone
electrophoresis using a charge-coupled devices with time delayed
integration," Anal Chem, 63, 496-502 (1991)). The most sensitive system
using a two dimensional CCD camera for the detection of fluorescent labels
was reported by Sweedler et al. Visible laser dyes are the main stay of
present systems, but infrared dye based systems (S. A. Soper, Q. L.
Mattingly and P. Vegnuta, "Photon burst detection of single near infrared
fluorescent molecules," Anal Chem, 65, pp. 740-747 (1993); S. A. Soper, &
Q. L. Mattingly, "Steady-state and picosecond laser fluorescence studies
of nonradiative pathways in tricarbocianine dyes: implication to the
design of near infrared fluorochromes with high fluorescence
efficiencies," I. Am. Chem. Soc., 116, pp. 3744-3752 (1993)) are also
commercially available (LICOR) sophisticated signal processing algorithms
are being developed for improving the base calling confidence; this
remains a hot issue, and many different approaches are being explored by
The optical system, which continues to use distributed bulk optics, such
as, microscope objectives, has not caught the imagination of many
researchers, yet if any significant inroads are to be made toward
increased throughput the optical system is a critical component in whole
set up. The standard optical system uses a microscope objective to
illuminate a small region in the flow cell, and a forward looking
microscope objective based detection system defines probe volume of 10-100
pl. The LICOR system has taken a first step toward improving the optical
system, their system combines the transmitting and receiving optics into a
compact unit, which is placed on the same side of the slab gel. The unit
is mounted onto a translational stage to allow scanning of several columns
in a short period of time. However, as the number of columns increases,
the increased scan time will cause two problems: firstly, the
signal-to-noise ratio will decrease, requiring a longer integration time
at each channel, which in turn will mean that data is not collected at the
same instant of time from all columns. Finally the LICOR system cannot
easily be adapted for use with a multi-capillary system. A fiber optic
system using axial illumination of a capillary has been reported by Taylor
and Yeung (1993), however, their system is invasive.
An improved optical system achieved by use of fiber optics, Dhadwahl, et
al. H. S. Dhadwal, R. R. Khan, and M. A. Dellavecchia, "A Coherent Fiber
Optic Sensor For Early Detection of Cataractogenesis in a Human Eye Lens,"
Optical Engineering: special issue on Biomedical Engineering, 32, pp.
223-238 (1993) ›Also published in Selected papers in Tissue Optics:
Application in Medical Diagnostic and Therapy, SPIE Milestone MS102
(1994)! and H. S. Dhadwal, K. Suh, and R. R. Khan, "Compact backscatter
fiber optic systems for submicroscopic particle sizing," Particulate
Science and Technology: An International Journal, 12, No. 2, pp. 139-148
(1994), is preferred to be used in connection with the present invention.
Optical fibers offer a unique alignment and motion free capability for
either multichannel capillary or slab gel based DNA sequencing systems.
FIG. 5 shows a schematic of a T-shape connector which links the receiving
and the transmitting fibers to an observation spot on a capillary. The
capillary is held in position using a miniature 3-chuck jaw assembly; the
fiber optical probe is positioned perpendicular to the capillary using the
special fixtures shown. These allow 3 degrees of freedom for the
The assembly contains fibers both for the excitation of fluorescence (fiber
transmitter) and the collection of the fluorescent response (fiber
receiver). It is proposed here that the fiber receivers be endowed with a
refractive index grating, so as to do discriminate between different
Thus, FIG. 6 depicts a fiber with a refractive index profile along the
fiber. This profile forms a set of one or more distributed Bragg
reflectors designed so as to reject the light of wavelengths corresponding
to the laser emission, .lambda..sub.1, .lambda..sub.2, and .lambda..sub.3.
The reflection spectrum of thus prepared fiber is depicted in the inset to
FIG. 7 depicts a fiber endowed with a refractive index profile,
corresponding to a Fabry-Perot resonator. Inset to FIG. 7 shows the
transmission spectrum of such a fiber. The Fabry-Perot fiber is designed
to select a desired wavelength of fluorescence, at .lambda..sub.0 in the
example depicted herein.
The choice of photodetector is determined by the fluorescent wavelength,
for example, avalanche photodiode (APDS) have much higher quantum
efficiencies at the near IR wavelength compared with photomultipliers.
APDS are also more compact and easy to operate, but do need to be cooled
to avoid non-linear gain effects due to self-heating. For visible
wavelength, a photomultiplier may be the detector of choice. These are
available in small packages which include built in high voltage biasing.
In either case, the optical signal is easily and reliably coupled to the
photodetector through the use of appropriate connectors.
The electronic read-out circuit which can be used in the present invention
processes the output of the photodetectors to determine the digital
equivalent of the fluorescence of the dyes, and can be used to transfer
the digital signal to a desktop computer.
The analog to digital (A/D) conversion and the data formatting can be
accomplished utilizing commercially available signal acquisition cards
installed in the expansion slots of desk-top computers.
The read-out circuit can be designed such that it can be interfaces
directly to common desktop computers through the system bus. A
programmable input/output device can be used to format the data and
interface to the computer. The card can be addressed by software similar
to any other input/output device in the system. The prototype systems will
be compatible with personal computers currently available.
Signal processing to estimate the sequence of nucleotide bases as
accurately as possible can be attained by hardware circuits and/or
software and software-assisted circuitry. These systems simultaneously
process four digital data records corresponding to the A, C, G, and T
bases, respectively. In addition to providing accurate calls, the methods
also produce confidence levels which reflect the probabilities of correct
Thus, while there have been described what are presently believed to be the
preferred embodiments of the present invention, those skilled in the art
will appreciate that modifications and changes made be made thereto
without departing from the true spirit of the invention, and it is
intended to include all such modifications and changes as come within the
scope of the claims which are appended hereto.
* * * * *