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BACKGROUND OF THE INVENTION
The capability for detecting microscopic particles has been proceeding
toward smaller particles. For many applications it is essential that
microscopic particles be detected in a liquid-phase environment. Existing
techniques, usable in a liquid-phase environment, are based on optical
trapping and on flow separation using hydrodynamically focused flows.
Molecular identification by laser-induced fluorescence has been used with
hydrodynamically focused flows to permit the detection of large and highly
fluorescent molecules using conventional photomultiplier tubes to detect
the molecule fluorescence.
Optical trapping and manipulation of viruses and bacteria are taught in A.
Ashkin et al., "Optical Trapping and Manipulation of Viruses and
Bacteria," Science 235, 1517 (1987). Rayleigh- and Mie-sized particles,
i.e., a particle size range from about 10 .mu.m down to a few angstroms,
have been trapped using optical forces to confine the particles. The only
method of identification taught by Ashkin et al. appears to be a size
determination from a scattering comparison with a sphere of known size.
Further, a large number of particles are trapped.
A hydrodynamically focused flow system is taught by D. C. Nguyen et al.,
"Ultrasensitive Laser-Induced Fluorescence Detection in Hydrodynamically
Focused Flows," J. Opt. Soc. Am. B4, 138 (1987), and D. C. Nguyen et al.,
"Detection of Single Molecules of Phycoerythrin in Hydrodynamically
Focused Flows by Laser Induced Fluorescence," Anal. Chem. 59, 2158 (1987),
incorporated herein by reference. As taught therein, improvements in the
optics and reductions in the size of the probe volume provide a
sensitivity effective to detect a single species containing the
fluorescence equivalent of eight rhodamine-6G chromophores. The detection
of single molecules of the highly fluorescent species phycoerythrin is
reported.
A variety of modifications are reported to enhance the detection
sensitivity of the device, with the improvements being related to
conventional optics and flow dynamics, and with a sample volume reduction
from 11 pL to 0.6 pL producing a concomitant reduction in detected
background radiation. The reported sensitivities do not, however, enable
the device to detect individual molecules that might typically be of
interest, such as fluorophore-tagged versions of the base molecules that
make up the DNA polymer.
Thus, available methods and apparatus for detecting particles in a flow
stream do not provide the sensitivity for detecting individual molecules
that might typically be encountered in immunofluorescence assay, flow
cytometry, liquid chromatography, and similar applications. An
agglomeration of molecules might be detected, but single molecules could
not then be identified. This lack of capabilities in the art is overcome
by the present invention and improved method and apparatus are provided
for detecting a single modestly fluorescent molecule.
Accordingly, it is an object of the present invention to reliably detect a
single fluorescent molecule.
Another object is to reliably detect single fluorescent molecules with a
fluorescence equivalent to flouorescently-labeled versions of the bases
forming the DNA polymer.
Yet another object is to provide an increased capability of rejecting
background radiation.
One other object is to minimize the resolution limitations inherent in
conventional optics while maintaining a large field of view.
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, the apparatus of this invention may comprise a molecule detection
system for identifying individual molecular characteristic emissions in a
train of molecules in a flow cell. A position sensitive sensor means is
located effective to detect emissions from molecules within the flow cell
and to assign spatial and temporal coordinates for the detected emissions.
A computer predicts spatial and temporal coordinates for a molecule in the
laminar flow as a function of the detected coordinates of a first detected
emission. Comparison means then compares detected spatial and temporal
coordinates with the predicted spatial and temporal coordinates to
determine whether a detected emission originated from an excited molecule
in the train of molecules. Thus, molecular emissions can be distinguished
from background emissions and identified with a particular molecule in the
sequence.
In another characterization of the present invention, a detection method is
provided for identifying individual molecules within a flow cell from
characteristic molecular emissions. Molecular emissions from within the
flow cell are detected with a position sensitive sensor. Spatial and
temporal coordinates are then assigned to the detected emissions. Based on
known flow characteristics in the flow cell, spatial and temporal
coordinates are predicted for a molecule in the flow as a function of a
first detected emission within the flow cell. The detected spatial and
temporal coordinates of subsequent emissions are compared with predicted
spatial and temporal coordinates to determine whether a detected emission
originated from a molecule in the train of molecules. Thus, molecular
emissions are distinguished from background events and a single molecule
can be identified during passage through the flow cell.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and form a part of the
specification, illustrate an embodiment of the present invention and,
together with the description, serve to explain the principles of the
invention. In the drawings:
FIG. 1 is a block diagram schematic of the present invention.
FIG. 2 is a detail of the system flow cell in pictorial form.
FIG. 3 is flow chart for distinguishing and evaluating individual molecules
in the flow cell.
DETAILED DESCRIPTION OF THE INVENTION
Referring now to FIG. 1, there is shown in block diagram schematic form a
molecule detection system according to the present invention. The laser
excitation system is generally well known and described in the Nguyen et
al. articles, hereinabove referenced. Laser 10 is selected with a
wavelength effective to fluoresce a selected fluorophore for identifying
the molecule to be detected. The output from laser 10 is conventionally
passed through half-wave plate 12 and polarizing prism 14, wherein the
output power of laser 10 can be adjusted by varying the angle of plate 12
with respect to prism 14. The laser output power and the polarization can
be adjusted to minimize background counts.
Mirror 16 directs the laser beam through lens 18 to focus within flow cell
22 for activating fluorophores attached to molecules in the sample stream.
As shown in FIG. 2, sample stream 42 is orthogonal to focused laser beam
46. Sample stream 42 passes within a surrounding hydraulic sheath 44 to
provide hydrodynamic focusing of the flow within flow cell 22.
Referring again to FIG. 1, the output from flow cell 22 is optical signal
20 with information on the fluorescing molecules within flow cell 22.
Optical signal 20 is focused by microscopic objective lens 24 and filtered
by spectral filter 25 to remove wavelengths which are not of interest. The
output optical signal is provided to a position-sensitive sensor 26. In
one embodiment, position-sensitive detector 26 is formed from a
microchannel plate position-sensitive detector (MCP) and operation is
hereinafter discussed with respect to a MCP.
A position-sensitive detector of MCP 26 outputs a signal which is
indicative of the occurrence of a photon event within flow cell 22 and
also location data functionally related to the spatial coordinates of the
photon event. Spatial coordinates are provided to digitizer 28 and
combined with a temporal input from timer 32 to provide at least a
three-dimensional (x, y, t) location for the photon event. Photon event
coordinates are output from digitizer 28 to memory 34 for subsequent
processing by computer station 36.
Referring now to position-sensitive sensor 26, it is desirable to have the
resolution of the system, and related position accuracy, limited by the
system optics rather than a MCP. Conventional MCPs may have a positional
resolution of 500-1000 pixels in each dimension. If two pixels cover each
Rayleigh limit, resolution is limited by the optics and a field of view of
100-200 microns in diameter is provided by objective lens 24. By way of
example, the Rayleigh limit at a wavelength of 560 nm is about 0.4
microns. Thus, a position accuracy of 1 micron requires only a precision
of .+-.2 pixels. A suitable MCP is available as model F4146M from ITT,
Electro-Optical Products Division.
Digitizer 28 provides spatial and temporal coordinate data in a format that
is suitable for direct storage in memory 34 and can operate in real time.
Conventional MCP position circuitry digitizes in about 5 .mu.s. This
digitizing interval can be reduced to about 1 .mu.s, or less, with custom
circuitry, if a high data rate operation is desirable. A 1 .mu.s
digitizing interval would enable a maximum photon detection rate of 1 MHz;
or, e.g., 170 photons during the transit time predicted by Nguyen et al.
for a system comparable to flow cell 22. As discussed below, the detection
of only a few photons can provide for reliable molecule identification
even with a relatively unsophisticated data reduction algorithm. The flow
velocity and laser intensity can readily be adjusted to provide a data
rate and observation time suitable for particular applications.
It will be appreciated that the above system provides the position accuracy
needed to identify a photon event within 1 micron or less. With a suitable
width of the laser beam in the longitudinal direction as, for example, by
evanescent wave illumination, this accuracy thus produces an effective
sample volume of 10.sup.-18 m.sup.3, or 10.sup.-3 l pL, a sample volume
reduction by about 500 over the 0.6 pL value discussed in Nguyen et al.
The effective sample volume allows the system to discriminate against
photon events which do not originate with a fluorescing molecule since
only a few scattering events will randomly occur within the effective
sample volume. The laminar flow provides known trajectories for molecules
having a known velocity. Detected photon events can be compared with
predicted molecule coordinates and photon events which do not correspond
with predicted coordinates can be disregarded. This capability effectively
provides a moving sample volume as small as 10.sup.-3 pL within which the
presence of a molecule can be reliably predicted.
The above system has been described using a hydrodynamic flow regime and a
fluorescing molecule. However, the functional principles apply equally to
any dynamic system which can maintain a predictable flow of molecules or
small particles in sequence through a detector. Likewise, laser-induced
fluorescence is a convenient technique to tag and identify molecules. All
that is needed, however, is a detectable emission from the molecule or
particle. Alternatively, other molecular emissions, such as electrons,
gamma rays, and the like, can be detected by suitable position-sensitive
devices. The present invention broadly contemplates hydrodynamic and
aerodynamic flow regimes, as well as molecular emissions of all kinds.
Referring now to FIG. 3, there is shown a flow diagram for exemplary
software for determining the presence of a molecule in an effective sample
volume. On the occurrence of a photon event 48 from MCP 16 (FIG. 1), the
event coordinates (x.sub.e, y.sub.e, t.sub.3) are input 52 and a bin is
defined 54 with coordinates x.sub.e, y.sub.e, t.sub.n) where t.sub.n is
normalized 53 to the time a molecule having the spatial coordinates would
have entered the field of view defined by objective lens 24 (FIG. 1). The
new defined bin is compared 56 with existing bins. If the new bin does not
exist, the new bin is stored 58 to represent an initial event and the
contents of bin file 58 are updated 62.
If existing bin coordinates accommodate the defined event bin coordinates
54, the event is assigned to that particular bin 64. Bins continue to
accumulate 64 events until the bin temporal coordinate indicate that the
bin has passed outside the field of view of the system.
Timer 66 periodically causes the bins to be examined 68 to determine
whether a bin is still within the field of view or whether a molecule was
present in that bin. Bins that accumulate a large number of events have a
higher probability of containing an actual molecule than bins with fewer
events. The small effective sample volume which is provided according to
the present invention can produce a clear separation between bins that
contain molecules and those that contain only random background events.
After the bin count is processed, the bin is cleared 74 for reuse. If the
presence of a molecule is indicated, the fluorescence data can be
processed 72 for the particular determination being made by the system.
More complex data reduction algorithms might further consider diffusion
and other departures from laminar flow that can occur in various
applications.
The capability to track and identify an individual molecule provides
applications which are not possible using conventional photomultiplier
tubes. In one important application, the system might be adapted to detect
and identify individual bases forming a DNA sequence. A plurality of laser
wavelengths, alone or in combination with separate filters 25 and
detectors 26 (FIG. 1), could be used to excite individual molecules as
they pass through the sample flow cell 22 to identify fluorescent
base-specific labels which are attached to the molecules. The track of a
molecule will alternately appear or disappear to enable molecule
identification during the excitation sequence. Several molecules may be
simultaneously present in flow cell 22 and be individually tracked for
identification.
While a single MCP system has been discussed above, it may be desirable to
provide two orthogonally placed MCPs to increase the number of photons
which are collected during transit of the molecule through flow cell 22
and to provide additional spatial information. Detected photon events
would be correlated to provide complete three-dimensional spatial
coordinates. A detected photon in each of two orthogonally placed
detectors will, in principle, enable a trajectory to be predicted, such
that the presence of a third photon on the computed four-dimensional
trajectory is evidence of a molecule passage.
It will be appreciated that the detection of these few photons in the
available transit times produces an infinitesimal probability of missing a
molecule entirely. By way of example, in a 2 MCP geometry, and a 170 .mu.s
transit time, shown by Nguyen et al., supra, in Table 3, it can be
estimated that each detector will accumulate 8 real and 40 background
photons. Thus, the 2 MCPs will detect a mean number of 16 photons,
providing a probability of less than 10.sup.-4 of detecting fewer than the
4 photons needed for molecule detection.
The software discussed for FIG. 3 can be provided for each MCP and the bins
merged during the processing. A merger determination could be made on the
basis of the available common information, i.e., time coordinates. Bins
might be examined for merging only after a minimum number of events are
accumulated in that bin, thereby assuring that the time coordinates of
both bins are sufficiently well determined to make a valid comparison for
the merger. The bin with the large number of accumulated events might be
selected for the merged bin, whereby all subsequent photon events on the
bin trajectory are assigned to a single bin.
The foregoing description of the preferred embodiment of the invention has
been presented for purposes of illustration and description. It 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 embodiment was 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.
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