U.S. patent number 6,049,380 [Application Number 09/169,025] was granted by the patent office on 2000-04-11 for single molecule identification using selected fluorescence characteristics.
This patent grant is currently assigned to Regents of the University of California. Invention is credited to Peter M. Goodwin, James H. Jett, Richard A. Keller, Nicholas P. Machara, Alan K. Van Orden.
United States Patent |
6,049,380 |
Goodwin , et al. |
April 11, 2000 |
Single molecule identification using selected fluorescence
characteristics
Abstract
Single fluorescent molecules in a flowing sample stream are
distinguished and identified using only a single laser excitation
wavelength. A sample stream is formed containing a dilute mixture
of single molecule fluorophores, wherein each one of the
fluorophores is serially ordered in the sample stream. The sample
stream is illuminated with s single excitation wavelength laser
effective to excite each fluorophore one at a time. Fluorescence
emission photons from each said fluorophore are detected. A burst
size is determined for each fluorophore to identify each
fluorophore. A pulsed laser may be used, where burst size and an
intra-burst fluorescence decay rate for each fluorophore are
determined simultaneously from the detected fluorescence emission
photons. The burst size and the decay rate are correlated to
identify each fluorophore.
Inventors: |
Goodwin; Peter M. (Los Alamos,
NM), Jett; James H. (Los Alamos, NM), Keller; Richard
A. (Los Alamos, NM), Van Orden; Alan K. (Los Alamos,
NM), Machara; Nicholas P. (Germantown, MD) |
Assignee: |
Regents of the University of
California (Los Alamos, MX)
|
Family
ID: |
26745527 |
Appl.
No.: |
09/169,025 |
Filed: |
October 9, 1998 |
Current U.S.
Class: |
356/317;
250/458.1 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 21/6408 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 021/64 (); G01J 003/30 ();
F21V 009/16 () |
Field of
Search: |
;356/317,318,72,73,417
;250/458.1,459.1,461.1,461.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Steven A. Soper, Lloyd M. Davis, and E. Brooks Shera, "Detection
and Identification of Single Molecules in Solution," J. Opt. Soc.
Am. B, vol. 9, No. 10, pp. 1761-1769, Oct. 1992. .
Peter M. Goodwin, Charles W. Wilkerson, Jr., W. Patrick Ambrose,
and Richard A. Keller, "Ultrasensitive Detection of Single
Molecules in Flowing Sample Streams by Laser-Induced Fluorescence."
SPIE, vol. 1895, Ultrasensitive Laboratory Diagnostics, pp. 79-89,
1993. .
Joel Tellinghulsen, W. Patrick Ambrose, John C. Martin, and Richard
A. Keller, "Analysis of Fluorescence Lifetime Data for Single
Rhodamine Molecules in Flowing Sample Streams," Anal. Chem., vol.
66, pp. 64-72, 1994. .
M. Sauer, K. T. Han, R. Muller, S. Nord, A Schulz, S. Seeger, J.
Wolfrum, J. Arden-Jacob, G. Deltau, N. J. Marx, C. Zander, and K.
H. Drexhage, "New Fluorescent Dyes in the Red Region for
Biodiagnostics," Journal of Fluorescence, vol. 5, No. 3, pp.
247-261, 1995. .
P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. N. Demas, J. H.
Jett, J. C. Martin, L. J. Reha-Krantz, D. J. Semin, J. A. Schecker,
M. Wu, and R. A. Keller, "Progress Toward DNA Sequencing at the
Single Molecule Level," Experimental Technique of Physics, vol. 41,
No. 2, pp. 279-294, 1995. .
Li-Qiang Li and Lloyd M. Davis, "Rapid and Efficient Detection of
Single Chromophore Molecules in Aqueous Solution," Applied Optics,
vol. 34, No. 18, pp. 3208-3216, Jun. 20, 1995. .
Peter M. Goodwin, W. Patrick Ambrose, and Richard A. Keller,
"Single-Molecule Detection in Liquids by Laser-Induced
Fluorescence," Accounts of Chemical Research, vol. 29, No. 12, pp.
607-613, 1996. .
C. Zander, M. Sauer, K. H. Drexhage, D. S. Ko, A. Schulz, J.
Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and
Characterization of Single Molecules in Aqueous Solution," Applied
Physics B, vol. 63, pp. 517-523, 1996. .
M. Kollner, A. Fischer, J. Arden-Jacob, K. H. Drexhage, R. Muller,
S. Seeger, and J. Wolfrum, "Fluorescence Pattern Recognition for
Ultrasensitive Molecule Identification: Comparison of Experimental
Data and Theoretical Approximations," Chemical Physics Letters,
vol. 250, pp. 355-360, 1996. .
Jorg Enderlein, Peter M. Goodwin, Alan Van Orden, W. Patrick
Ambrose, Rainer Erdmann, and Richard A. Keller, "A Maximum
Likelihood Estimator to Distinguish Single Molecules by Their
Fluorescence Decays," Chemical Physics Letters, vol. 270, pp.
464-470, 1997. .
M. Sauer, C. Zander, R. Muller, B. Ullrich, K. H. Drexhage, S.
Kaul, and J. Wolfrum, "Detection and Identification of Individual
Antigen Molecules in Human Serum with Pulsed Semiconductor Lasers,"
Applied Physics B Lasers and Optics, vol. 65, Issue 3, pp. 427-431,
1997. .
Alan Van Orden, Nicholas P. Machara, Peter M. Goodwin, and Richard
A. Keller, "Single-Molecule Identification in Flowing Sample
Streams by Fluorescence Burst Size and Intraburst Fluorescence
Decay Rate," Analytical Chemistry, vol. 70, No. 7, pp. 1444-1451,
Apr. 1, 1998. .
C. Eggeling, J. R. Fries, L. Brand, R. Gunther, and C. A. M.
Siedel, "Monitoring Conformational Dynamics of a Single Molecule by
Selective Fluorescence Spectroscopy," National Academy of Sciences,
vol. 95, pp. 1556-1561, 1998. .
Markus Sauer, Jutta Arden-Jacob, Karl H. Drexhage, Florian Gobel,
Ulrike Lieberwirth, Klaus Muhlegger, Ralph Muller, Jurgen Wolfrum,
and Christoph Zander, "Time-Resolved Identification of Individual
Mononucleotide Molecules in Aqueous Solution with Pulsed
Semiconductor Lasers," Bioimaging, vol. 6, pp. 14-24,
1998..
|
Primary Examiner: Font; Frank G.
Assistant Examiner: Lauchman; Layla
Attorney, Agent or Firm: Wilson; Ray G.
Parent Case Text
RELATED CASES
This application claims the benefit of the priority date of
provisional application Ser. No. 60/065,365, filed Nov. 12, 1997.
Claims
What is claimed is:
1. A method for identifying single fluorescent molecules in a
sample stream comprising the steps of:
forming a sample stream containing a dilute mixture of selected
single molecule fluorophores, wherein each one of said fluorophores
is serially ordered in said sample stream;
passing said serially ordered fluorophores one at a time through a
detection volume;
illuminating said sample stream within said detection volume with a
pulsed laser beam at a single excitation wavelength effective to
excite said fluorophores;
detecting emitted photons from each one of said fluorophores to
output corresponding photon detection events;
determining from said photon detection events a photon burst size
and an intra-burst fluorescence decay rate for each one of said
fluorophores; and
correlating said burst size and said decay rate with known
characteristics for each one of said fluorophores to identify each
one of said fluorophores.
2. A method according to claim 1, wherein said sample stream has a
diameter less than a diameter of said laser beam intercepting said
sample stream.
3. A method according to claim 1, wherein the step of forming said
sample stream includes the step of directing a sheath fluid about
said sample stream to form a focused sample stream within said
laser beam.
4. A method according to claim 1, further including the steps
of:
photobleaching said sheath fluid; and
introducing said dilute mixture of single molecule fluorophores
within said sheath fluid after said photobleaching.
5. A method according to claim 1, further including the step of
forming a photon record comprising a number of said photon
detection events occurring within a time-gated window after a
predetermined delay with respect to an exciting pulse in said
pulsed laser beam and a detection time for each one of said photon
detection events that is relative to an immediately preceding
photon detection event.
6. A method according to claim 1, wherein the step of determining
said size of each said photon burst further includes the step
of:
time filtering each said photon burst to output filtered burst data
comprising a number of said photon detection events.
7. A method according to claim 1, wherein the step of determining
said intra-burst fluorescence decay rate further includes the step
of estimating said intra-burst fluorescence decay rate from arrival
times of said photons measured with respect to said excitation
laser pulses.
8. A method according to claim 1, wherein said fluorophores are
selected to maximize the resolution between said fluorophores in
burst size and fluorescence decay rate space.
9. A method for identifying single fluorescent molecules comprising
the steps of:
forming a sample stream containing a dilute mixture of selected
single molecule fluorophores, wherein each one of said fluorophores
is serially ordered in said sample stream;
passing said serially ordered fluorophores one at a time through a
detection volume;
illuminating said sample stream within said detection volume with a
laser beam at a single excitation wavelength effective to excite
said fluorophores;
detecting emitted photons from each said fluorophore to output
corresponding photon detection events;
determining from said photon detection events a photon burst size
for each one of said fluorophores; and
correlating said burst size with known characteristics for each one
of said fluorophores to identify each one of said fluorophores.
10. A method according to claim 9, wherein said sample stream has a
diameter less than a diameter of said laser beam intercepting said
sample stream.
11. A method according to claim 9, wherein the step of forming said
sample stream includes the step of directing a sheath fluid about
said sample stream to form a focused sample stream within said
laser beam.
12. A method according to claim 9, further including the steps
of:
photobleaching said sheath fluid; and
introducing said dilute mixture of said single molecule
fluorophores within said sheath fluid after said
photobleaching.
13. A method according to claim 9, wherein the step of determining
said size of said photon bursts further includes the step of:
time filtering each said photon burst to output filtered burst data
comprising a number of said photon detection events.
14. A method according to claim 9, wherein said fluorophores are
selected to maximize the resolution between said fluorophores in
burst size space.
15. A method according to claim 9, wherein said laser beam is a
pulsed laser beam.
16. A method according to claim 15, further including the step of
forming a photon record comprising a number of said photon
detection events occurring within a gated window after a
predetermined delay with respect to an exciting pulse in said
pulsed laser beam and a detection time for each one of said photon
detection events that is relative to an immediately preceding
photon detection event.
17. A method according to claim 16, wherein the step of determining
said size of said photon bursts further includes the step of:
time filtering each said photon burst to output filtered burst data
comprising a number of said photon detection events.
18. A method according to claim 16, wherein said fluorophores are
selected to maximize the resolution between said fluorophores in
burst size space.
Description
BACKGROUND OF THE INVENTION
This invention relates to flow cytometry and, more particularly, to
applications of flow cytometry using single molecule
identification. This invention was made with government support
under Contract No. W-7405-ENG-36 awarded by the U.S. Department of
Energy. The government has certain rights in the invention.
Light-induced fluorescence detection of single molecules in liquid
solution was first accomplished several years ago, and applications
for single molecule detection (SMD) in the analytical,
environmental, and biomedical sciences are beginning to emerge.
Overviews of single molecule detection in solution are presented
in, e.g., R. A. Keller et al., 50 Appl. Spectrosc., pp. A12-A32
(1996), and P. M. Goodwin et al., 29 Accounts Chem. Res., pp.
607-613 (1996). In general, an operable system will include a
dilute stream of separated individual molecules with known
fluorescent characteristics that are excited one at a time by a
light source, where the resulting emitted photons are detected. One
approach to single molecule detection used herein is based on flow
cytometry, where the analyte solution is delivered into a rapidly
flowing sheath fluid and hydrodynamically focused into a narrow
sample stream. Yet another approach is based on analyte movement
through a capillary, such as described in U.S. Pat. No. 5,209,834,
issued May 11, 1993, to Shera, and incorporated herein by
reference.
In flow cytometry, a sample stream passes through the center of a
probe volume defined by the diameter of the focused excitation
laser beam and a spatial filter placed in the image plane of a
light collecting objective. Single fluorescent molecules are
detected by the bursts of photons emitted as they flow through the
detection volume one-at-a-time. Hydrodynamic focusing of the sample
stream by the sheath fluid ensures that the entire sample stream
passes through the center of the excitation laser so that single
molecules delivered into the flow cell are detected with an
efficiency exceeding 90%. See, e.g., P. M. Goodwin et al.,
"Progress toward DNA sequencing at the single molecule level," 41
Exp. Tech. Phys., pp. 279-294 (1995), incorporated herein by
reference.
Some of the applications under development for efficient single
molecule detection in flow include DNA fragment sizing, DNA
sequencing, counting and sorting of single molecules, and detection
of probe-target binding. A number of applications for SMD in
solution require one to distinguish between different fluorophores
present in a mixture. For example, in one approach to DNA
sequencing (U.S. Pat. No. 4,962,037, issued Oct. 9, 1990, and
incorporated herein by reference), each base is labeled with a
different fluorescent probe, and a rapid, efficient method is
needed to identify these fluorophores. In yet another approach,
only two fluorescent probes are required (U.S. Pat. No. 5,405,747,
issued Apr. 11, 1995, and incorporated herein by reference) to
reduce the number of distinguishing characteristics that are
required to be identified.
Several techniques have been developed to distinguish between
different single molecules in solution. One technique employs two
or more detection channels to identify single molecules in a
multicomponent mixture based upon differences in excitation and
emission wavelengths of the fluorophore labels. See, e.g., Soper et
al., "Detection and identification of single molecules in
solution," 9 J. Opt. Soc. Am. B, pp.1761-1769 (October 1992),
incorporated herein by reference. Typically, the detection volume
is probed by collinear laser beams at different wavelengths
corresponding to the excitation maxima of each of the fluorophores.
A beam splitter directs the fluorescence from each fluorophore to a
separate filter/detector channel. This method requires that the
emission bands of the fluorophores be sufficiently separated to
minimize crosstalk between the respective channels. Furthermore,
separating the fluorescence signal into distinct detection channels
increases the complexity of the instrumentation and can reduce the
overall detection efficiency.
An alternative approach that requires only a single detection
channel and is applicable to molecules with similar spectroscopic
properties exploits the difference in fluorescence lifetimes of the
fluorophores. Single Rhodamine 6G (R6G) molecules in flow have been
distinguished from Rhodamine B (RB) molecules and from tetramethyl
rhodamine isothiocyanate (TRITC) molecules by applying a maximum
likelihood function to time-correlated single-photon counting
(TCSPC) fluorescence decay measurements of individual bursts in a
mixed sample (Zander et al.,63 Appl. Phys. B, pp. 517-523 (1996);
J. Enderlein et al., 270 Chem. Phys. Lett., pp. 464-470 (1997)).
Also, a rhodamine derivative JA169 has been distinguished from a
carbocyanine dye Cy5 using this technique (M. Sauer et al., 65
Appl. Phys. B. No. 3, pp. 427-431 (August 1997).
Objects, advantages and novel features of the invention will be set
forth in part in the description which follows, and in part will
become apparent to those skilled in the art upon examination of the
following or may be learned by practice of the invention. The
objects and advantages of the invention may be realized and
attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with
the purposes of the present invention, as embodied and broadly
described herein, this invention comprises a method for identifying
single fluorescent molecules in a flowing sample stream. A sample
stream is formed containing a dilute mixture of single molecule
fluorophores, wherein each one of the fluorophores is serially
ordered in the sample stream. The sample stream is illuminated with
a single excitation wavelength laser beam effective to excite each
fluorophore. Emitted photons from each said fluorophore are
detected. In one embodiment, a burst size is determined for each
fluorophore to identify each fluorophore. In another embodiment,
the laser beam is formed of pulses, and a burst size and an
intra-burst fluorescence decay rate (.GAMMA.) for each fluorophore
are determined from fluorescence emission photons. The burst size
and the decay rate are correlated to identify each fluorophore.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and form a
part of the specification, illustrate the embodiments of the
present invention and, together with the description, serve to
explain the principles of the invention. In the drawings:
FIG. 1 schematically depicts apparatus for identifying single
fluorescent molecules.
FIGS. 2A-2D graphically depict single molecule fluorescence photon
burst data from a photolysed ultrapure water blank, a dilute sample
stream of TRITC, a dilute sample stream of R6G, and a dilute sample
stream with a mixture of TRITC and R6G, respectively.
FIGS. 3A and 3B graphically depict single molecule fluorescence
burst duration distributions measured from dilute sample streams of
TRITC and R6G, respectively.
FIGS. 4A-4F graphically depict fluorescence photon burst size
distributions (BSDs) compiled from data collected from a dilute
sample stream of R6G, of TRITC and a mixture of R6G and TRITC
(FIGS. 4A, 4C, 4E), and corresponding BSDs that are time filtered
(FIGS. 4B, 4D, 4F).
FIGS. 5A and 5F are correlated single molecule burst size and
intraburst fluorescence decay rate (.GAMMA.) measurements for a
sample stream containing approximately equal amounts of TRITC and
R6G (FIGS. 5A-C) and a sample stream containing mostly R6G (FIGS.
5D-F).
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, single molecule
identification (SMI) is based on differences in detected
fluorescence burst intensities from fluorescent molecules. These
intensity differences are a consequence of the different
photophysical properties (absorption cross-sections, fluorescence
quantum yields, and fluorescence emission spectra) of different
fluorescent molecules. Unlike discrimination methods based on
separate excitation wavelengths and emission detection channels or
by fluorescence decay rates, burst intensity discrimination
requires that the molecules experience a similar excitation
irradiance during their transit across an excitation laser beam.
The excitation laser beam may be either a pulsed laser beam or a
continuous-wave laser beam. To accomplish this, the molecules are
constrained to a sample stream diameter within the focused
excitation laser. As with fluorescence decay rate measurements,
this technique can be applied to molecules with similar
spectroscopic properties and requires only a single excitation
wavelength and fluorescence emission detection channel. In
addition, a multiplex technique, wherein individual fluorescent
molecules are distinguished by simultaneous measurement of
fluorescence burst intensity and intra-burst fluorescence decay
rate, provides a relatively high probability of individual
fluorescent molecule identification. When intra-burst fluorescence
decay rate is determined, the excitation laser beam must be a
pulsed laser beam.
EXPERIMENTAL SECTION
Single molecule detection (SMD) in a flowing sample stream has been
detailed in several publications, as noted in the Background of the
Invention. The experimental techniques and results presented herein
are also reported in Van Orden et al., "Single-Molecule
Identification in Flowing Sample Streams by Fluorescence Burst Size
and lntraburst Fluorescence Decay Rate," 70 Anal. Chem. No. 7, pp.
1444-1451 (Apr. 1, 1998), incorporated herein by reference. An
operable system requires only a sample stream having an ordered
flow of molecules that pass through the center of an excitation and
detection volume. The experimental apparatus described herein
provides increased efficiency for the detection of single molecules
and is a preferred apparatus, but the present invention is not
limited to flow cytometry. Other devices are available that provide
an ordered stream of molecules, e.g., capillary flow and
microchannels, and are known to persons of ordinary skill in the
detection of single molecules and are within the scope of the
present invention.
To obtain the results reported below, apparatus similar to that
schematically shown in FIG. 1 was used. The parameters specified
below are exemplary only and not intended to limit the scope of the
present invention. Solutions 12 of, e.g., R6G and TRITC fluorophore
molecules dissolved in ultrapure water, were delivered 14 into a
sheath fluid 16, flowing through a square bore flow cell 18
(250.times.250 .mu.m.sup.2), from fused silica capillary 22 (10
.mu.m i.d., 140 .mu.m o.d.). The outlet tip 24 of the capillary was
etched to a narrow taper using hydrofluoric acid to facilitate
smooth laminar flow of the sheath fluid around the capillary. The
inlet end 26 of sample introduction capillary 22 was positioned
.about.20 cm above outlet 28. A vacuum regulator (not shown) was
used to apply a partial vacuum on the inlet end of the capillary to
control the sample introduction rate. This configuration resulted
in a sample stream diameter of approximately 10 .mu.m. Sheath fluid
16 was ultrapure water delivered to the top of flow cell 18 by pump
32, which is a syringe pump in the experimental embodiment, but
could be any controllable fluid delivery system, such as gravity
feed, a positive displacement pump, or a roller-type pump. A second
pump 34, which was also a syringe pump in the experimental
embodiment, was used to move sheath fluid 16 through flow cell 18
at a volumetric flow rate of nominally 25-50
.mu..lambda.min.sup.-1.
Fluorescent impurities present in sheath fluid 16 were photolyzed
by passing the sheath fluid through a .about.1 m-long photolysis
cell 36 before it entered the flow cell, as described in U.S. Pat.
application Ser. No. 08/727,841, incorporated herein by reference.
Fluorescence was induced by irradiating the sample stream .about.25
.mu.m downstream from the capillary tip with a 514.5 nm mode-locked
argon ion laser beam 38 (82 MHz repetition rate, 200 ps pulse
width, 20-30 mW average power) focused to a circular spot of 16
.mu.m (e.sup.-2) diameter by a 75 mm focal length lens 42. A
60.times., 1.2 NA water immersion microscope objective 44 was used
to collect emitted fluorescence photons along an axis orthogonal to
the flow and excitation axes. A 600 .mu.m-wide, 1000 .mu.m -high
slit (not shown), with the long axis oriented parallel to the flow
axis, was placed in the image plane of the collection objective to
limit the size of the detection volume to .about.2 p .pi.. Emitted
fluorescence photons were converted to photoelectrons by
conventional detection electronics 48 and recorded as photon
detection events.
Hydrodynamic focusing of the sample stream by the sheath fluid to a
diameter of .about.10 .mu.m ensured that the entire sample stream
flowed through the central portion of the detection volume. When
this is the case, SMD efficiencies >90%, essentially limited by
the photostability of the fluorescent species, are possible. Light
passing through the slit was spectrally filtered with a bandpass
filter (575.+-.15 nm) (not shown) and focused onto a single photon
counting avalanche photodiode within detection electronics 48 using
a 32.times.microscope objective. Time-correlated single photon
counting (TCSPC) was used to measure the elapsed time between the
excitation laser pulse and each detected photon. Background due to
Raman and Rayleigh scattering of the excitation laser beam by the
solvent was suppressed by rejecting photon counts detected within
.about.1 ns of the laser pulse.
DATA ANALYSIS
Each detected photon arriving with a delay >1 ns with respect to
the excitation laser pulse (gated photon) was recorded. The record
consists of detection times (with respect to the previous gated
photon) for each gated photon as well as the arrival time of the
photon with respect to the excitation laser pulse. A photon burst
is evidenced by a series of successive gated photons recorded at a
high rate (40-100 kHz) compared to the background counting rate
(.about.5 kHz). Details of the TCSPC apparatus and the algorithm
used to search data sets for photon bursts are described in, e.g.,
P. M. Goodwin et al., 1895 P. Soc. Photo-opt Ins., pp. 79-89
(1993), incorporated herein by reference.
Here, burst search threshold times of 75 and 50 .mu.s were used for
data collected with 20 and 30 mW of average excitation laser power,
respectively. Successive gated photons (photoelectrons) detected at
time intervals less than the threshold time comprise a photon
burst. Each detected photon burst is characterized by three
parameters: (1) the number of photoelectrons (PE) comprising the
burst (burst size); (2) the duration of the burst (accumulated time
below threshold); and (3) the intra-burst fluorescence decay rate
(.GAMMA.). The intra-burst fluorescence decay rate is the
reciprocal of the fluorescence lifetime .tau..sub.f, where
.GAMMA.=1/.lambda..sub.f. While the decay rate is the preferred
parameter for the present invention, it is intended herein that the
use of fluorescence decay rates include the use of fluorescence
lifetimes, if desired by an experimenter. The intra-burst
fluorescence decay rate was estimated from the distribution of
intra-burst photon arrival times measured with respect to the
excitation laser pulse, using a maximum likelihood estimator for a
background free, single exponential decay (J. Tellinghuisen et al.,
"Analysis of Fluorescence Lifetime Data for Single Rhodamine
Molecules in Flowing Sample Streams," 66 Anal. Chem., pp. 64-72
(1994), incorporated herein by reference). To reduce contributions
due to background bursts and accidental coincidences (a
fluorescence burst recorded with two or more analyte molecules in
the detection volume simultaneously), bursts were time-filtered,
that is, bursts with durations significantly shorter or longer (see
below) than the mean molecular transit time across the detection
volume were discarded.
The following procedure was used to estimate the probabilities for
misidentification of the two fluorophores and to characterize the
most probable sources of error. A Monte Carlo (MC) simulation was
used to generate fluorescence burst data from a simulated flow
cytometry-based SMD experiment, given the photophysical properties
of the analyte molecules (absorption cross section, fluorescence
quantum yield, fluorescence lifetime, photodestruction quantum
yield, optical saturation intensity), analyte fluorescence emission
detection efficiencies of the apparatus, excitation laser
parameters (pulse repetition rate, average power, focused spot
size), and flow parameters (analyte concentration, sample stream
flow velocity). The simulation accounts for spatial variations of
the excitation laser intensity and fluorescence collection
efficiency within the detection volume, as well as diffusion,
photobleaching, and optical saturation of the analyte molecules
during their transit through the detection volume. Good agreement
between the simulation and the experimental data was achieved by
making slight adjustments to the estimated optical
collection/detection efficiency of the apparatus. For the R6G
simulation, it was also necessary to adjust the photodestruction
efficiency, as further discussed in Van Orden et al.,supra.
P. M. Goodwin, et al., 1895 P. Soc. Photo-op Ins., supra.,
describes the application of a Monte Carlo simulation to model the
fluorescence burst size distribution detected from single
Rhodamine-110 molecules excited in flow. The simulation has been
modified to model the TCSPC data of the simulated bursts as well as
the fluorescence burst sizes. For each detected photon, a random
deviate drawn from the appropriate TCSPC arrival time distribution
(R6G, TRITC, or background fluorescence) was used to assign a time
separation between that detected photon and the excitation laser
pulse. The background photon arrival time distribution was compiled
directly from the experimental data by excluding photons detected
inside of photon bursts. R6G and TRITC fluorescence photon arrival
time distributions were constructed by multiplication of single
exponential decays (4.0 and 2.3 ns for R6G and TRITC, respectively,
taken from bulk TCSPC measurements of these dyes) by the TCSPC
photon arrival distribution measured from an uncorrelated light
source (ideally, a flat distribution) to account for nonlinearities
of the TCSPC system.
RESULTS AND DISCUSSION
For all data sets presented in FIGS. 2A-D, 3A-B, and 4A-F, the
average excitation laser power was 20 mW. The sheath fluid
volumetric flow rate was 40 .mu..lambda. min.sup.-1 ; the e.sup.-2
transit time across the detection volume, derived from the
autocorrelation function of the photon burst data, was 1.2 ms; and
the velocity of the sample stream through the detection volume,
estimated from the MC simulation, was 1.2 cm/sec.
FIGS. 2A-D display 500 ms of raw fluorescence burst data binned at
250 .mu.s intervals from a photolyzed ultrapure water blank (FIG.
2A) and dilute sample streams of TRITC (FIG. 2B), R6G (FIG. 2C),
and a mixture of R6G and TRITC (FIG. 2D). A comparison of FIGS. 2B
and 2C clearly shows that the fluorescence burst sizes detected for
single TRITC molecules are, on average, smaller than those detected
for R6G As discussed above, the fluorescence burst size of a single
fluorophore is determined by such photophysical properties as the
absorption cross section at the excitation wavelength, the
fluorescence quantum yield, the fluorescence lifetime, the overlap
of the fluorescence emissions, and the spectral bandwidth of the
detection channel. For TRITC, the peak in the absorption spectrum
occurs near 554 nm. Therefore, excitation at 514.5 nm does not
occur as efficiently as for R6G, with an absorption maximum at 528
nm. Furthermore, the fluorescence quantum yield of R6G (0.9) is
almost three times that of TRITC (0.35). To improve the relative
fluorescence emission detection efficiency for TRITC, a spectral
bandpass filter was used in the detection channel that favored the
fluorescence emission maximum of TRITC.
FIGS. 3A and 3B display fluorescence burst duration distributions
(BDD) compiled from data collected from dilute sample streams of
TRITC (FIG. 3A) and R6G (FIG. 3B). Experimental conditions were the
same as for the data shown in FIGS. 2A-D. BDDs compiled from data
collected with the sample stream ON are shown with solid circles;
BDDs collected with the sample stream OFF and misaligned with
respect to the detection volume are shown with open circles. The
peak in each distribution gives the mean detected burst duration
for unphotobleached TRITC and R6G molecules under these
experimental conditions. Note that the mean detected burst
durations are significantly shorter than the e.sup.-2 transit time
(1.2 ms) derived from the autocorrelation function. This is a
consequence of the burst search algorithm threshold. The majority
of single molecule fluorescence bursts fall in a time range
(0.42-1.38 ms) denoted by the vertical lines and arrows in FIGS. 3A
and 3B. Shorter bursts are primarily due to photoelectrons
associated with Raman scattering that "leak" through the time
gates, and longer bursts are mostly accidental coincidences. Thus,
a time-filter can be selected to discriminate against background
emissions and accidental coincidence bursts in the data in
real-time during data collection or, as is done here, in a
post-processing step.
FIGS. 4A-F display size distributions of emitted fluorescence
photon bursts compiled from data collected under the same
conditions as for FIGS. 2A-D and 3A-B. Burst size distributions
(BSD) shown with closed circles in FIGS. 4A, 4C, and 4E were
compiled from data collected from dilute sample streams of R6G,
TRITC, and a R6G/TRITC mixture, respectively. Open circles are BSDs
compiled from the water blank (data collected with the sample
stream OFF and misaligned with respect to the detection
volume).
FIGS. 4B, 4D, and 4F, display BSDs that were obtained after the
burst data was time-filtered to remove bursts less than 0.42 ms and
greater than 1.38 ms in duration. This time-filtering procedure was
done to reduce the number of background bursts (<0.42 ms) and
bursts resulting from two or more molecules simultaneously present
(>1.38 ms) in the detection volume from the data set. Note the
marked reduction in the number of smaller (<20 PE) bursts
primarily due to background emissions. It must be emphasized that
the background BSDs were not subtracted from the analyte BSDs to
obtain the time-filtered distributions shown in FIGS. 4B, 4D, and
4F.
The peaks in the BSDs at .about.30 and .about.60 PE correspond to
the average fluorescence burst sizes detected from single TRITC and
R6G molecules, respectively, that did not photobleach while
crossing the detection volume. The fact that peaks are observed in
the BSDs indicates that the diameter of the sample stream was
indeed smaller than the detection volume and was well aligned to
the laser beam. Were this not the case, the BSD would simply
decrease monotonically away from zero PE, i.e., the BSD is a
maximum at 0 PE.
FIGS. 4E and 4F show raw and time-filtered BSDs obtained from the
mixture of TRITC and R6G. Two well-resolved peaks at 30 and 60 PE
are observed, corresponding to different BSDs for TRITC and R6G,
respectively. This result confirms that these fluorophores can be
distinguished at the single molecule level based on their different
fluorescence burst sizes. The solid curves in FIGS. 4A-4F are BSDs
obtained from synthetic data generated by the MC simulation
described above. The simulation parameters were adjusted to give
the best fits to the mixture in FIGS. 4E and 4F. These same
parameters were then used for the simulations of the pure R6G and
TRITC solutions.
Table 1 presents the parameters used in the simulation. Asterisks
indicate parameters that were adjusted to achieve the best fit. A
time-filtered burst size detection threshold of 14 PE was chosen as
a good compromise between background burst rate and SMD efficiency.
Time-filtered bursts smaller than 14 PE in size are not counted as
single molecule fluorescence bursts. For this threshold, the
simulation indicates that 96% of the TRITC molecules and 78% of the
R6G molecules that are introduced into the sheath flow are detected
by apparatus used herein. The smaller efficiency obtained for R6G
was due to the larger photodestruction quantum yield for this
molecule.
TABLE 1 ______________________________________ Monte Carlo
Simulation Parameters Parameter R6G TRITC
______________________________________ Absorption cross section
.sigma..sub.514nm (10.sup.-16 cm.sup.2) 2.2 1.6 Fluorescence
quantum yield 0.9 0.35 Fluorescence lifetime (ns) 4.0 2.3
Photodestruction quantum yield (10.sup.-6) 80* 6 Saturation
intensity (10.sup.5 W cm.sup.-2 s.sup.-1) 0.55 1.14 Diffusion
constant (10.sup.-8 cm.sup.2 s.sup.-1) 300 300 Bandpass filter
fluorescence emission 0.16 0.27 transmission Time-gate fluorescence
emission transmission 0.84 0.74 Excitation laser .theta..sup.-2
beam diameter (10.sup.-4 16) 16 Averge excitation laser power
(10.sup.-3 W) 20.sup.+, 30.sup.++ 20.sup.+, 30.sup.++ Sample flow
velocity (cm s.sup.-1) 1.23.sup.+, 1.23.sup.+, 0.9.sup.++
0.9.sup.++ Overall optical collection/detection efficiency
0.093*.sup.+, 0.093*.sup.+, excluding bandpass filter and time gate
0.085*.sup.++ 0.085*.sup.++ transmissions Detection electronics
dead time (10.sup.-6) 4 4 ______________________________________
*values adjusted to fit experimental data .sup.+ For data in FIGS.
2-4; .sup.++ For data in FIG. 5
In order to fit the R6G burst size distribution (FIG. 3A), the
photodestruction quantum efficiency of R6G was adjusted to a final
value of 8.times.10.sup.-5. This value is larger than the value of
2.times.10.sup.-5 reported in the literature. In FIG. 4B, a
shoulder is observed toward the low photoelectron side of the peak.
This shoulder is due to R6G molecules that photobleached during
their transit through the detection volume. A much smaller shoulder
is seen for the TRITC peak in FIG. 4D due to the higher
photostability of TRITC. The simulation herein indicates that 44%
of the detected R6G molecules photobleach while crossing the
detection volume compared to 4% of the TRITC molecules.
A single molecule identification (SMI) threshold of 45 PE was
chosen; bursts greater than or equal to this threshold are
identified as R6G single molecule fluorescence bursts and bursts
<45 PE are identified as TRITC bursts. According to the MC
simulation, 75% of detected R6G single molecule bursts are
.gtoreq.45 PE in size, and 25% are detected below this threshold.
Of the detected TRITC single molecule bursts, 96% are <45 PE in
size and 4% give bursts .gtoreq.45 PE; 97% of the unphotobleached
R6G molecules are detected with bursts .gtoreq.45 PE. Clearly,
photodestruction of R6G molecules as they transit the detection
volume increases error rates for SMI based on fluorescence burst
size alone. But, as indicated by FIGS. 4E and 4F, increasing the
photostability of the brighter component will reduce the overlap of
the burst size distributions and decrease the SMI error rates.
To improve the accuracy of SMI, simultaneous, correlated
measurements of the burst size and intra-burst fluorescence decay
rate were performed for each detected burst in R6GITRITC mixtures.
For these measurements, the flow velocity of the sample stream
through the detection volume was lowered to 0.9 cm s.sup.-1 and the
average excitation power was raised to 30 mW to increase the
average number of PE detected per burst.
FIGS. 5A and 5B show correlated burst size (PE) and fluorescence
decay rate (.GAMMA.) measurements from dilute sample streams
containing approximately equal amounts of TRITC and R6G (FIG. 5A)
and containing mainly R6G (FIG. 5B). Analyte molecule introduction
rates estimated from MC simulations are 20 TRITC s.sup.-1 and
.about.20 R6G s.sup.-1 in FIG. 5A; and .about.65 R6G s.sup.-1 and
.about.3 TRITC s.sup.-1 in FIG. 5B. The correlated data are
displayed as two-dimensional histograms (scatter plots) with darker
shades of grey indicating increasing numbers of events.
Experimental conditions were: a sheath volumetric flow rate, 30
.mu..lambda. min.sup.-1 ; average excitation laser power, 30 mW;
sample stream flow velocity, .about.0.9 cm s.sup.-1 ; and sample
transit time (e.sup.-2), .about.1.8 ms. The burst data were
time-filtered (0.25-2.0 ms) to reduce background bursts and
accidental coincidences; only bursts .gtoreq.20 PE in size are
shown. Again, no background subtraction was done to arrive at the
distributions shown in the figures.
The PE-F plane was divided into four regions (I,II,III,IV) for SMI,
as shown in FIGS. 5A and 5B. Projections of the correlated data
onto the fluorescence decay rate and fluorescence burst size axes
are shown to the left and above each scatter plot, respectively.
Solid curves A and B plotted on the projections are estimates,
based on MC simulations of the experiment, of TRITC and R6G
contributions to the measured distributions, respectively. Curves C
are sums of the estimated TRITC and R6G contributions. Vertical
lines shown on the projections delimit regions that are used for
distinguishing TRITC and R6G single molecule fluorescence bursts
based on intra-burst fluorescence decay rate
alone(TRITC.ident.I+IV, .GAMMA..gtoreq.0.307 ns.sup.-1 ;
R6G.ident.II+III, .GAMMA.<0.307 ns.sup.-1) and burst size
(TRITC.ident.I+III, 20.ltoreq.PE<75; R6G.ident.II+IV,
PE.gtoreq.75).
Based on MC simulations for these experimental conditions and a
time-filtered burst size detection threshold of .gtoreq.20 PE, the
estimated SMD efficiencies are 97% for TRITC and 80% for R6G.
Again, the lower detection efficiency for R6G is due to its high
photodestruction quantum yield; approximately 66% of the R6G
molecules photobleach while crossing the excitation laser, whereas
only 7% of the TRITC molecules photobleach. The increased
photobleaching rates (compared to FIGS. 4A-F) are due to the higher
average excitation laser power (1.5.times.) and longer sample
transit time (1.4.times.) used in this experiment.
According to the MC simulations, for SMI by burst size alone, 99%
of the detected TRITC molecules give bursts .gtoreq.20 and <75
PE in size and fall into Region I+III; only 1% of the detected
TRITC molecules give bursts >75 PE and fall into Region II+IV.
53% of the detected R6G molecules give bursts .gtoreq.75 PE and
fall into Region II+IV, and 47% of the detected R6G molecules give
bursts >20 and <75 PE in size and fall into Region I+III.
Again, a large fraction of the detected R6G molecules are
misidentified. This is a consequence of detected bursts from
photobleached R6G molecules that fall into the size range (I+III)
expected for TRITC bursts.
For SMI by intra-burst fluorescence decay rate (.GAMMA.) alone, 89%
of the detected TRITC molecules give bursts with
.GAMMA..gtoreq.0.307 ns.sup.-1 and 11 % give bursts with
.GAMMA.<0.307 ns.sup.-1. For R6G.sub.1 88% of the detected
molecules give bursts with .GAMMA.<0.307 ns.sup.-1 and 12% give
bursts with .GAMMA..gtoreq.0.307 ns.sup.-1. Here the errors are a
consequence of the overlap of the estimated fluorescence decay rate
distributions for TRITC and R6G. The widths of these distributions
are determined by the number of PE used to estimate the decay rate.
Improvement of the overall photon collection/detection efficiency
would result in more PEs detected per burst and narrower
fluorescence decay rate distributions, thereby increasing the
accuracy of SMI. Photobleaching does broaden the R6G distribution
indirectly since, on average, fewer PEs are used to estimate
fluorescence decay rate of photobleached R6G molecules. However,
this effect is small compared to the direct effect that
photobleaching has on the R6G burst size distribution.
For correlated, two-parameter SMI, two islands corresponding to
fluorescence bursts detected from unphotobleached TRITC and R6G
molecules are clearly visible in FIGS. 5A-C. TRITC bursts with a
mean burst size of 53 PE and a mean intra-burst fluorescence decay
rate of 0.38 ns.sup.-1 cluster in Region I; unphotobleached R6G
bursts with a mean burst size of 96 PE and a mean intra-burst
fluorescence decay rate of 0.25 ns.sup.-1 cluster in Region II.
Region III contains mostly bursts from R6G molecules that
photobleached while crossing the detection volume. This is clear
from FIGS. 5D-F where the sample consisted mainly of R6G.
Photobleached R6G molecule fluorescence bursts comprise a "tail" on
the unphotobleached R6G distribution that extends from Region II
across Region III.
Based on the MC simulation, probabilities are assigned for detected
TRITC and R6G bursts to fall into Regions I-IV. These are tabulated
in Table 2.
TABLE 2 ______________________________________ Single Molecule
Identification Probabilities I II III IV
______________________________________ TRITC 0.883 0.001 0.108
0.008 R6G 0.078 0.489 0.390 0.044
______________________________________
Probabilities for detection in regions delineated for SMI by burst
size (I+III,II+IV) and intra-burst fluorescence decay rate
(I+IV,II+III) alone were obtained by summing the appropriate
entries in Table 2. These calculations ignore accidental
coincidences and are strictly valid only for SMD rates small
compared to the reciprocal of the molecular transit time across the
detection volume.
It is interesting to calculate the confidence level for
identification as R6G or TRITC for events that occur in the
specific regions. This level depends on the relative detection
rates of the two molecules. For example, for SMI by burst size
alone, the magnitude of the tail of the TRITC BSD extending into
Region II+IV depends on the detection rate of TRITC. Likewise the
magnitude of the shoulder of the R6G BSD into Region I+III depends
on the detection rate of R6G.
The fraction of bursts falling into a given region due to TRITC or
R6G can be calculated from the relative detection rates for these
species and the SMI probabilities given in Table 2. Table 3 is a
tabulation of the fraction of bursts detected in a region due to a
specific fluorophore for three different sample stream
concentration ratios of TRITC and R6G ([TRITC]/[R6G]=0.25, 1, 4).
For example, comparison of the results for an equimolar sample
shows that the highest SMI confidences are obtained for the
correlated measurement in Regions I (93% of the bursts detected in
this region are due to TRITC) and II (99.8% for R6G). SMI by burst
size alone gives only a 72% confidence for TRITC (Region I+III) and
a 98% confidence for R6G (Region II+IV). SMI by intra-burst
fluorescence decay rate alone gives a 90% identification confidence
for TRITC (Region I+IV) and a 87% confidence for R6G (Region
II+III).
TABLE 3 ______________________________________ Single Molecule
Identification Confidence Levels
______________________________________ 1 Parameter - Burst Size
TRITC.vertline.R6G I + III II + IV
______________________________________ 0.25 TRITC 0.39 0.006 R6G
0.61 0.994 1.0 TRITC 0.72 0.02 R6G 0.28 0.98 4.0 TRITC 0.91 0.08
R6G 0.09 0.92 ______________________________________ 1 Parameter:
Fluorescence Decay Rate TRITC.vertline.R6G I + IV II + III
______________________________________ 0.25 TRITC 0.69 0.04 R6G
0.31 0.96 1.0 TRITC 0.90 0.13 R6G 0.10 0.87 4.0 TRITC 0.97 0.38 R6G
0.03 0.62 ______________________________________ 2 Parameter: Burst
Size + Fluorescence Decay Rate TRITC.vertline.R6G I II III IV
______________________________________ 0.25 TRITC 0.77 0.001 0.08
0.05 R6G 0.23 0.999 0.92 0.95 1.0 TRITC 0.93 0.002 0.25 0.18 R6G
0.07 0.998 0.75 0.82 4.0 TRITC 0.98 0.01 0.57 0.47 R6G 0.02 0.99
0.43 0.53 ______________________________________
CONCLUSIONS
Thus, in accordance with the present invention, single molecules in
mixed sample streams may be distinguished with high accuracy based
on differences in fluorescence burst intensity or a combination of
fluorescence burst intensity and intra-burst fluorescence decay
rate, both using only a single excitation wavelength and a single
fluorescence emission detection channel. For the analytes studied
here, TRITC and R6G, SMI confidence levels for TRITC are limited
mainly by the photostability of R6G. Increasing the photostability
of the analytes by, for example, the addition of an anti-fade
reagent is expected to result in significant improvement of SMI
confidence levels.
An immediate application is to single molecule DNA sequencing.
Sequencing multiple identical DNA strands individually and
combining the results to form a consensus sequence will reduce
error rates below those reported here. For example, given a random
error for sequencing an individual DNA strand of 20% per base, the
calculations show that the error for a consensus sequence having
only 10 DNA strands is reduced to less than 1%.
While the above examples are based on the pair of dyes Rhodamine 6G
and TRITC, many other dye selections can be identified readily by a
person skilled in the art of fluorescence detection. Since only one
excitation wavelength and a single detection channel are used, the
various fluorescent species chosen for SMI must have overlapping
excitation and overlapping emission spectra. The excitation
wavelength and emission bandpass filter wavelength must be chosen
such that the fluorescent species can both be detected at the
single molecule level with photon bursts of sufficient size
(greater than about 25 detected photons per burst) for intra-burst
fluorescence decay measurements to be obtained.
In the absence of optical saturation and photobleaching, the
intensity of photon bursts from a given species scales as the
product of the absorption cross-section and the fluorescence
quantum yield of the molecule. Photobleaching will reduce the
average burst size and broaden the burst size distribution. Both
the excitation wavelength and detection channel bandpass filter
center position and width can be adjusted to optimize differences
between the average detected photon burst intensities of the
different fluorescent species.
The fluorescence decay rate of a given species is determined by its
photophysical properties and interactions between it and its
immediate environment (e.g., the solvent). The chosen species must
have fluorescence decay rates sufficiently different such that they
can be discriminated at the single molecule (photon burst) level.
Given the current state of the art for overall photon
collection/detection efficiencies (1%-5%), the decay rate ratio of
the chosen species should be at least 1.5 for SMI based on lifetime
alone. This ratio is expected to decrease as detection technology
improves. Differences in the species' average photon burst
intensities allows SMI with smaller decay rate ratios (closer to
unity).
In general, for single molecule identification by fluorescence
burst size or by a combination of fluorescent burst size and
intra-burst fluorescence decay rate, a condition is selected to
maximize the resolution between the species in the burst size and
burst size/fluorescence decay rate plane. SMD efficiencies and SMI
error rates for a given pair of dyes can be predicted using a MC
simulation that incorporates photophysical constants of the dyes
and selected experimental parameters. Some possible fluorescent dye
pairs for SMI according to the above criteria are provided in Table
4.
TABLE 4 ______________________________________ Dye Combinations for
SMI abs/em abs/em Dye 1 (nm) .GAMMA. (ns).sup.-1 Dye 2 (nm) .GAMMA.
(ns).sup.-1 ______________________________________ Lisamine 570/590
0.475 Texas Red 595/615 0.250 Rhodamine B Rhodamine B 570/590 0.556
Oregon 511/530 0.238 Green 514 TRITC 555/580 0.475 BODIPY FI
505/513 0.175 R6G 528/555 0.263 TRITC 555/580 0.475 Cresyl Violet
610/620 0.313 BODIPY TR 589/617 0.185 Perchlorate TRITC 555/580
0.475 Oregon 503/522 0.244 Green 500 Rhodamine B 570/590 0.556
Oregon 503/522 0.244 Green 500 TRITC 555/580 0.475 Rhodamine
570/590 0.231 101 Perchlorate Rhodamine 652/678 0.475 Rhodamine
682/716 0.91 700 800 Cresyl Violet 610/620 0.313 Rhodamine 570/590
0.556 Perchlorate B Cy 5 655/675 0.665 Cresyl 610/620 0.313 Violet
Perchlorate ______________________________________
The foregoing description of the invention has been presented for
purposes of illustration and description and is not intended to be
exhaustive or to limit the invention to the precise form disclosed,
and obviously many modifications and variations are possible in
light of the above teaching. The embodiments were chosen and
described in order to best explain the principles of the invention
and its practical application to thereby enable others skilled in
the art to best utilize the invention in various embodiments and
with various modifications as are suited to the particular use
contemplated. It is intended that the scope of the invention be
defined by the claims appended hereto.
* * * * *