U.S. patent application number 10/953677 was filed with the patent office on 2005-02-24 for system and method for providing improved event reading and data processing capabilities in flow cytometer.
Invention is credited to Brown, Scott, Helms, Steven, Hopkins, Perry, Payavala, Sreedhar, Stokdijk, Willem, Yount, Dwayne.
Application Number | 20050042760 10/953677 |
Document ID | / |
Family ID | 33163236 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050042760 |
Kind Code |
A1 |
Yount, Dwayne ; et
al. |
February 24, 2005 |
System and method for providing improved event reading and data
processing capabilities in flow cytometer
Abstract
A system and method for use with a flow cytometer to improve
event reading and data processing capabilities of the flow
cytometer, while also providing efficient system configuration
assessment capabilities. The system and method enables the flow
cytometer to capture and sample an entire waveform representative
of an event being read, and provides improved processing and
analysis of the sampled data in a real time or near real-time
basis. The system and method further enable the flow cytometer to
assess its configuration and provide assessment results to an
operator in an efficient and effective manner.
Inventors: |
Yount, Dwayne; (Campbell,
CA) ; Brown, Scott; (Santa Cruz, CA) ;
Payavala, Sreedhar; (San Jose, CA) ; Stokdijk,
Willem; (Livermore, CA) ; Hopkins, Perry;
(Fremont, CA) ; Helms, Steven; (Scottsdale,
AZ) |
Correspondence
Address: |
DAVID W. HIGHET, VP AND CHIEF IP COUNSEL
BECTON, DICKINSON AND COMPANY
1 BECTON DRIVE, MC 110
FRANKLIN LAKES
NJ
07417-1880
US
|
Family ID: |
33163236 |
Appl. No.: |
10/953677 |
Filed: |
September 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10953677 |
Sep 29, 2004 |
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09853043 |
May 11, 2001 |
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6809804 |
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60203515 |
May 11, 2000 |
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60203590 |
May 11, 2000 |
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60203585 |
May 11, 2000 |
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60203577 |
May 11, 2000 |
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Current U.S.
Class: |
436/63 ;
422/73 |
Current CPC
Class: |
G06K 9/6218 20130101;
G01N 2015/1477 20130101; G01N 15/1459 20130101; G01N 2015/1006
20130101 |
Class at
Publication: |
436/063 ;
422/073 |
International
Class: |
G01N 033/48 |
Claims
What is claimed is:
1. A system for processing at least one signal representative of an
event detected by at least one detector in a flow cytometer, the
system comprising: a sampling device, adapted to receive portions
of said signal from said detector in time sequence and to generate
a respective value representative of the respective magnitude of
each respective portion of said signal as said respective portion
of said signal is being received; and a storage device, adapted to
store said values generated by said sampling device.
2. A system as claimed in claim 1, wherein: said sampling device
receives a number of said portions totaling substantially all of
said signal, and generates said values which represent said
portions of substantially all of said signal.
3. A system as claimed in claim 1, wherein: said signal is an
analog signal representative of a light signal emitted from said
event as detected by said detector.
4. A system as claimed in claim 1, further comprising: an
arithmetic device, adapted to arithmetically combine a designated
value with each of said values.
5. A system as claimed in claim 4, wherein: said arithmetic device
includes a subtractor which is adapted to subtract said designated
value from each of said values.
6. A system as claimed in claim 4, wherein: said designated value
is representative of an undesired signal detected by said
detector.
7. A system as claimed in claim 4, wherein: said designated value
is representative of a characteristic of said detector.
8. A system as claimed in claim 1, wherein: said sampling device is
adapted to receive portions of a second said signal from a second
said detector in time sequence and to generate a respective second
value representative of the respective magnitude of each respective
portion of said second signal as said respective portion of said
second signal is being received; and said storage device is adapted
to store said second values generated by said sampling device.
9. A system as claimed in claim 8, wherein: said sampling device
receives said portions of said signal at a time different from that
during which said sampling device receives at least some of said
portions of said second signal.
10. A system as claimed in claim 9, further comprising: a
comparator, adapted to compare each of said second values with a
respective one of said first values to compare said signal to said
second signal.
11. A system for identifying a configuration of a detector unit of
a flow cytometer, the system comprising: a port, adapted to couple
to a removable device, said removable device including an optical
element and a memory adapted to store information pertaining to
said optical element; and a reader, adapted to read said
information stored in said memory when said removable device is
coupled to said port.
12. A system as claimed in claim 11, wherein: said optical element
includes an optical filter.
13. A system as claimed in claim 11, wherein: said optical element
includes a mirror.
14. A system as claimed in claim 11, further comprising: an
indicator, adapted to provide an indication of said information
read by said reader.
15. A removable device, adapted for coupling with a port of a flow
cytometer, the removable device comprising: an optical element; and
a memory adapted to store information pertaining to said optical
element.
16. A removable device as claimed in claim 15, wherein: said
optical element includes an optical filter.
17. A removable device as claimed in claim 15, wherein: said
optical element includes a mirror.
18. A method for processing at least one signal representative of
an event detected by at least one detector in a flow cytometer, the
method comprising: receiving portions of said signal from said
detector in time sequence; generating a respective value
representative of the respective magnitude of each respective
portion of said signal as said respective portion of said signal is
being received; and storing said values.
19. A method as claimed in claim 18, wherein: said receiving
receives a number of said portions totaling substantially all of
said signal; and said generating generates said values which
represent said portions of substantially all of said signal.
20. A method as claimed in claim 18, wherein: said signal is an
analog signal representative of a light signal emitted from said
event as detected by said detector.
21. A method as claimed in claim 18, further comprising:
arithmetically combining a designated value with each of said
values.
22. A method as claimed in claim 21, wherein: said arithmetic
combining includes subtracting said designated value from each of
said values.
23. A method as claimed in claim 21, wherein: said designated value
is representative of an undesired signal detected by said
detector.
24. A method as claimed in claim 21, wherein: said designated value
is representative of a characteristic of said detector.
25. A method as claimed in claim 18, further comprising: receiving
portions of a second said signal from a second said detector in
time sequence; generating a respective second value representative
of the respective magnitude of each respective portion of said
second signal as said respective portion of said second signal is
being received; and storing said second values.
26. A method as claimed in claim 25, wherein: said receiving steps
are performed such that said portions of said signal are received
at a time different from that during which at least some of said
portions of said second signal are received.
27. A method as claimed in claim 26, further comprising: comparing
each of said second values with a respective one of said first
values to compare said signal to said second signal.
28. A method for identifying a configuration of a detector unit of
a flow cytometer, comprising: coupling a removable device to a port
of said flow cytometer, said removable device including an optical
element and a memory adapted to store information pertaining to
said optical element; and reading said information stored in said
memory when said removable device is coupled to said port.
29. A method as claimed in claim 28, wherein: said optical element
includes an optical filter.
30. A method as claimed in claim 28, wherein: said optical element
includes a mirror.
31. A method as claimed in claim 28, further comprising: providing
an indication of said information read from said memory.
32. A method for manufacturing a removable device, adapted for
coupling with a port of a flow cytometer, the method comprising:
coupling an optical element to said removable device; and including
a memory in said removable device, said memory being adapted to
store information pertaining to said optical element.
33. A method as claimed in claim 32, wherein: said optical element
includes an optical filter.
34. A method as claimed in claim 32, wherein: said optical element
includes a mirror.
Description
[0001] The present invention claims benefit under 35 U.S.C. .sctn.
119(e) of a U.S. Provisional Patent Application of Dwayne Yount et
al. entitled "Hardware and Electronics Architecture for a Flow
Cytometer", Ser. No. 60/203,515, filed May 11, 2000, of a U.S.
Provisional Patent Application of Michael Lock et al. entitled
"Cluster Finder Algorithm for Flow Cytometer", Ser. No. 60/203,590,
filed May 11, 2000, of a U.S. Provisional Patent Application of
Michael Goldberg et al. entitled "User Interface and Network
Architecture for Flow Cytometer", Ser. No. 60/203,585, filed May
11, 2000, and of a U.S. Provisional Patent Application of John
Cardott et al. entitled "Digital Flow Cytometer", Ser. No.
60/203,577, filed May 11, 2000, the entire contents of each of said
provisional patent applications being incorporated herein by
reference.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] Related subject matter is disclosed in a copending U.S.
patent application of Pierce 0. Norton entitled "Apparatus and
Method for Verifying Drop Delay in a Flow Cytometer", Ser. No.
09/346,692, filed Jul. 2, 1999, in a copending U.S. patent
application of Kenneth F. Uffenheimer et al. entitled "Apparatus
and Method for Processing Sample Materials Contained in a Plurality
of Sample Tubes", Ser. No. 09/447,804, filed Nov. 23, 1999, and in
a copending U.S. patent application of Michael D. Lock et al.
entitled "System for Identifying Clusters in Scatter Plots Using
Smoothed Polygons with Optimal Boundaries", Attorney Docket No.
P-5100, filed even date herewith, the entire contents of each of
these applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of The Invention
[0004] The present invention relates to a system and method for
providing improved event reading, data processing and system
configuration capabilities in a flow cytometer. In particular, the
present invention provides a system and method for use with a flow
cytometer that enables the event reading components of the flow
cytometer to capture and digitize substantially the entire optical
waveform of each detected event, and provides improved, near
real-time processing of the digitized waveform data and automated
system configuration assessment capabilities.
[0005] 2. Description of the Related Art
[0006] Flow cytometers known in the art are used for analyzing and
sorting particles in a fluid sample, such as cells of a blood
sample or particles of interest in any other type of biological or
chemical sample. A flow cytometer typically includes a sample
reservoir for receiving a fluid sample, such as a blood sample, and
a sheath reservoir containing a sheath fluid. The flow cytometer
transports the particles (hereinafter called "cells") in the fluid
sample as a cell stream to a flow cell, while also directing the
sheath fluid to the flow cell.
[0007] Within the flow cell, a liquid sheath is formed around the
cell stream to impart a substantially uniform velocity on the cell
stream. The flow cell hydrodynamically focuses the cells within the
stream to pass through the center of a laser beam. The point at
which the cells intersect the laser beam, commonly known as the
interrogation point, can be inside or outside the flow cell. As a
cell moves through the interrogation point, it causes the laser
light to scatter. The laser light also excites components in the
cell stream that have fluorescent properties, such as fluorescent
markers that have been added to the fluid sample and adhered to
certain cells of interest, or fluorescent beads mixed into the
stream.
[0008] The flow cytometer further includes an appropriate detection
system consisting of photomultipliers tubes, photodiodes or other
light detecting devices, which are focused at the intersection
point. The flow cytometer analyzes the detected light to measure
physical and fluorescent properties of the cell. The flow cytometer
can further sort the cells based on these measured properties.
[0009] To sort cells by an electrostatic method, the desired cell
must be contained within an electrically charged droplet. To
produce droplets, the flow cell is rapidly vibrated by an acoustic
device, such as a piezoelectric element. The droplets form after
the cell stream exits the flow cell and at a distance downstream
from the interrogation point. Hence, a time delay exists from when
the cell is at the interrogation point until the cell reaches the
actual break-off point of the droplet. The magnitude of the time
delay is a function of the manner in which the flow cell is
vibrated to produce the droplets, and generally can be manually
adjusted, if necessary.
[0010] To charge the droplet, the flow cell includes a charging
element whose electrical potential can be rapidly changed. Due to
the time delay which occurs while the cell travels from the
interrogation point to the droplet break-off point, the flow
cytometer must invoke a delay period between when the cell is
detected to when the electrical potential is applied to the
charging element. This charging delay is commonly referred to as
the "drop delay", and should coincide with the travel time delay
for the cell between the interrogation point and the droplet
break-off point to insure that the cell of interest is in the
droplet being charged.
[0011] At the instant the desired cell is in the droplet just
breaking away from the cell stream, the charging element is brought
up to the appropriate potential, thereby causing the droplet to
isolate the charge once it is broken off from the stream. The
electrostatic potential from the charging circuit cycles between
different potentials to appropriately charge each droplet as it is
broken off from the cell stream.
[0012] Because the cell stream exits the flow cell in a
substantially downward vertical direction, the droplets also
propagate in that direction after they are formed. To sort the
charged droplet containing the desired cell, the flow cytometer
includes two or more deflection plates held at a constant
electrical potential difference. The deflection plates form an
electrostatic field which deflects the trajectory of charged
droplets from that of uncharged droplets as they pass through the
electrostatic field. Positively charged droplets are attracted by
the negative plate and repelled by the positive plate, while
negatively charged droplets are attracted to the positive plate and
repelled by the negative plate. The lengths of the deflection
plates are small enough so that the droplets which are traveling at
high velocity clear the electrostatic field before striking the
plates. Accordingly, the droplets and the cells contained therein
can be collected in appropriate collection vessels downstream of
the plates.
[0013] Known flow cytometers similar to the type described above
are described, for example, in U.S. Pat. Nos. 3,960,449, 4,347,935,
4,667,830, 5,464,581, 5,483,469, 5,602,039, 5,643,796 and
5,700,692, the entire contents of each patent being incorporated by
reference herein. Other types of known flow cytometer, are the
FACSVantage.TM., FACSort.TM., FACSCount.TM., FACScan.TM. and
FACSCalibur.TM. systems, each manufactured by Becton Dickinson and
Company, the assignee of the present invention.
[0014] Although the flow cytometers described above can be suitable
for reading events as intended, these existing systems do suffer
from certain drawbacks. For example, in these types of instruments,
the controller or central processing unit (CPU) does not ordinarily
process the data obtained from reading the events in "real time".
However, it is desirable to process the data in real time or near
real time to improve the efficiency of the flow cytometer and the
ability to compare the readings of the events on a real-time or
near real-time basis.
[0015] These existing systems also do not capture the entire image
of the event. That is, when each event is read by detecting, for
example, light fluorescing from the cell or particle of interest,
these systems capture the "peak point" or peak intensity of the
detected light. These systems also typically measure the duration
during which the light is detected. By detecting these two
parameters, the existing systems can use this data to determine
characteristics of the event, such as the identity and size of a
cell of interest. However, these techniques do not enable the
existing systems to sample individual regions of the cell or
particle of interest, nor are they capable of being performed on a
real-time or near real-time basis. Furthermore, these systems are
typically incapable of comparing data from multiple events
effectively and in a real time or near real-time manner.
[0016] In addition, these types of existing systems do not provide
a mechanism that indicates the configuration of the system to the
operator effectively. For example, these types of systems are
typically configured with multiple detector and filter arrangements
that enable the different detectors to detect light having
wavelengths within different wavelength regions. In such an
arrangement, one detector can detect light with having a wavelength
within the range of blue light, for example, while another detector
can detect light having a wavelength within the range of green
light. However, if an incorrect filter is placed in front of a
particular detector, the detector will detect the incorrect light
(e.g., green light instead of blue light). The system will
therefore give erroneous results. However, the operator of the
system will have difficulty determining which filters are arranged
incorrectly, and in the worst case, the error may go unnoticed.
[0017] Accordingly, a need exists for an improved system and method
for use with a flow cytometer to improve the event reading and data
processing features of the flow cytometer to eliminate the above
drawbacks.
SUMMARY OF THE INVENTION
[0018] An object of the present invention is to provide a system
and method for use with a flow cytometer to improve event reading
and data processing capabilities of the flow cytometer, while also
providing efficient system configuration assessment
capabilities.
[0019] Another object of the present invention is to provide a
system and method that enables a flow cytometer to capture and
sample an entire waveform representative of an event being read,
and which provides improved processing and analysis of the sampled
data in a real-time or near real-time basis.
[0020] A further object of the present invention is to provide a
system and method that is capable of indicating the configuration
of a flow cytometer to an operator in an efficient and effective
manner.
[0021] These and other objects are substantially achieved by
providing a system and method for processing at least one signal
representative of an event detected by at least one detector in a
flow cytometer. The system and method employs a sampling device
which is adapted to receive portions of the signal from the
detector in time sequence and to generate a respective value
representative of the respective magnitude of each respective
portion of the signal as the respective portion of the signal is
being received. The system and method further employ a storage
device which is adapted to store the values generated by the
sampling device. The sampling device can receive substantially all
of the signal, and can generate the values which represent the
portions of substantially all of the signal. The signal can be an
analog signal representative of a light signal emitted from the
event as detected by the detector. The system and method can
further employ an arithmetic device which is adapted to, for
example, subtract a designated value from each of the values
generated by the sampling device. The designated value can be
representative of an unwanted signal, such as crosstalk, detected
by the detector, or can be representative of a characteristic of
the detector. The sampling device can further be adapted to receive
portions of a second signal from a second detector in time sequence
and to generate a respective second value representative of the
respective magnitude of each respective portion of the second
signal as the respective portion of the second signal is being
received, and the storage device can store the second values
generated by the sampling device. The sampling device can receive
the portions of the signal at a time different from that during
which it receives at least some of the portions of the second
signal, and the system and method can employ a comparator which is
adapted to compare each of the second values with a respective one
of the values to compare the signal to the second signal.
[0022] These and other objects are further substantially achieved
by providing a system and for identifying a configuration of a
detector unit of a flow cytometer. The system and method employ a
port which is adapted to couple to a removable device that includes
an optical element, such as a mirror or filter, and a memory
adapted to store information pertaining to the optical element. The
system and method further employ a reader which is adapted to read
the information stored in the memory when the removable device is
coupled to the port. The system and method can also employ an
indicator which adapted to provide an indication of the information
read by the reader.
[0023] These and other objects are also substantially achieved by
providing a removable device which is adapted for coupling with a
port of a flow cytometer, and comprises an optical element, such as
a filter or mirror, and a memory adapted to store information
pertaining to the optical element.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The various objects, advantages and novel features of the
present invention will now be more readily appreciated from the
following detailed description when read in conjunction with the
accompanying drawings, in which:
[0025] FIG. 1 is a conceptual block diagram of the flow cytometer
employing a system and method according to an embodiment of the
present invention;
[0026] FIG. 2 is a cross-sectional view of the flow cytometer shown
in FIG. 1;
[0027] FIG. 3 is a detailed view of an example of an emission block
according to an embodiment of the present invention which is
employed in the flow cytometer shown in FIGS. 1 and 2;
[0028] FIG. 4 is a top perspective view of an example of a support
ring and flex circuits employed in the emission block shown in FIG.
3;
[0029] FIG. 5 is a bottom perspective view of the support ring and
flex circuits shown in FIG. 4;
[0030] FIG. 6 is a side view of the support ring and flex circuits
shown in FIGS. 4 and 5;
[0031] FIG. 7 is a conceptual top plan view of the emission block
shown in FIG. 3;
[0032] FIG. 8 is a perspective view of an example of a removable
mirror assembly for use with the emission block shown in FIG. 3 in
accordance with an embodiment of the present invention;
[0033] FIG. 9 is a perspective view of an example of a removable
mirror assembly for use with the emission block shown in FIG. 3 in
accordance with an embodiment of the present invention;
[0034] FIG. 10 is a conceptual top view of the emission block shown
in FIG. 3 illustrating exemplary paths in which light entering the
emission block is reflected and propagates;
[0035] FIG. 11 is a block diagram illustrating an example of the
electronic components employed in the flow cytometer shown in FIGS.
1 and 2 according to an embodiment of the present invention;
[0036] FIG. 12 is a block diagram illustrating anther example of
the electronic components employed in the flow cytometer shown in
FIGS. 1 and 2 according to another embodiment of the present
invention;
[0037] FIGS. 13-16 are conceptual illustrations of an exemplary
relationship between multiple lasers and multiple emission blocks
in the flow cytometer shown in FIGS. 1 and 2 according to an
embodiment of the present invention;
[0038] FIGS. 17-20 are conceptual block diagrams showing exemplary
relationship between certain components shown in FIGS. 11 and
12;
[0039] FIG. 21 illustrates an example of a waveform as captured and
sampled by the circuitry shown in FIGS. 11 and 12;
[0040] FIG. 22 is a conceptual block diagram of control circuitry
for a PMT detector; and
[0041] FIGS. 23-27 illustrate exemplary waveforms and their
processing by the circuitry shown in FIGS. 11 and 12.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] A flow cytometer 100 employing an embodiment of the present
invention is illustrated in FIGS. 1 and 2. As discussed in the
background section above, the flow cytometer 100 includes a nozzle
102 having a flow cell 104 therein. The flow cytometer further
includes a sample reservoir 106 for receiving a fluid sample,. such
as a blood sample, and a sheath reservoir 108 containing a sheath
fluid. The flow cytometer transports the cells in the fluid sample
in the cell stream to the flow cell 104, while also directing the
sheath fluid to the flow cell 104.
[0043] Within the flow cell 104, the sheath fluid surrounds the
cell stream, and the combined sheath fluid and cell stream exits
the flow cell 104 via an opening 110 as a sample stream. The
opening 110 can have a diameter of, for example, 50 .mu.m, 70
.mu.m, 100 .mu.m, or any other suitable diameter. As illustrated,
due to characteristics of the sheath fluid, such as surface tension
and the like, the sample stream remains intact until breaking off
into droplets at the droplet break-off point 112, which is at a
certain distance from opening 110. The distance from opening 110 at
which the droplet break-off point 112 occurs, and the frequency or
rate at which the droplets are formed, are governed by the fluid
pressure, as well as the amplitude and frequency of oscillation of
oscillating device 114 which can be, for example, a piezoelectric
element.
[0044] As shown in FIG. 2, the oscillating device 114 is connected
to an alternating voltage source 116 whose output voltage
amplitude, frequency and phase is controlled by a controller 118
which can include, for example, a microprocessor or any other
suitable controlling device. Further details of the controller 118
are described below. The amplitude of the alternating voltage
signal output by alternating voltage source 116 can be increased or
decreased by controller 118 to in turn increase or decrease the
distance from opening 110 at which the droplet break-off 112
occurs. Likewise, the frequency of the alternating voltage signal
output by alternating voltage source 116 can be increased or
decreased by controller 118 to increase or decrease the rate at
which droplets of sample fluid are formed at the droplet break-off
point 112.
[0045] To view the droplet break-off point 112, a light source 119,
such an LED array, can be positioned in the region of the sample
fluid stream containing the droplet break-off point 112. The
controller 118 can control the light source 119 to strobe at a
described frequency, so that the detector 120, such as a camera or
other special viewing device, can be used to view the region of the
sample fluid stream containing the droplet break-off point 112. The
flow cytometer 100 further includes at least one laser 122, such as
a diode laser, which is controlled by controller 118 to emit laser
light. The emitted laser light intersects the sample stream at a
point of interest 124 commonly referred to as a the interrogation
point.
[0046] The laser 122 can be, for example, a red laser that emits
light having a wavelength of at or about 633 nm, which is in the
red light spectrum. Alteratively, laser 122 can be a blue laser
that emits light having a wavelength of at or about 488 nm, which
is in the blue light spectrum. Laser 122 also can be an ultraviolet
laser that emits light having a wavelength of at or about 325 nm,
or within the range of at or about 351 nm to at or about 364 nm,
all of which are within the ultraviolet spectrum. As discussed in
more detail below, flow cytometer 100 can include multiple lasers
122 that each emit their respective laser light to a respective
interrogation point along the fluid flow stream. Also, if desired,
a lens or filter 126 can be positioned between the laser 122 and
the interrogation point 124 to filter out light of unwanted
wavelengths from the laser light prior to its reaching the
interrogation point 124.
[0047] As further illustrated, the flow cytometer includes at least
one fiberoptic cable 130 that receives laser light that has
intersected the sample stream at the interrogation point 124 and
has been scattered by the sample stream fluid and, in particular,
by any cells or particles of interest present in the sample stream.
The input port 132 of the fiberoptic cable 130 in this example is
located in the same plane as the laser light being emitted from
laser 122, and at a 90.degree. angle or about a 90.degree. angle
with respect to the direction of propagation of the laser light
being emitted from laser 122. The laser light scattered by the
fluid stream and any cells or particles of interest at the
interrogation point 124 is commonly referred to as side-scatter
laser light.
[0048] As further illustrated, a detector 134 and filter 136
arrangement can be used to detect a portion of the laser light that
has passed through the interrogation point 124 along the direction
of propagation of the laser light being emitted by laser 122, which
is commonly referred to as the forward-scatter laser light. Also,
if desired, an obscuration bar 138 can be position in the path of
the forward-scatter laser light, in the path of the side-scatter
laser light, or in both paths, to reduce the amount of side-scatter
laser light entering fiber optic cable 130 or to reduce the amount
of forward-scatter laser light entering detector 134. The
side-scatter laser light entering the fiberoptic cable 130 is input
to an emission block 140 as described in more detail below.
[0049] As further shown in FIGS. 1 and 2, the flow cytometer 100
can include deflection plates 142 and 144 which can be controlled
by controller 118 to allow droplets to pass to droplet collection
container 146, or to deflect droplets that have been charged by
charging unit 147 towards droplet collection containers 148 and
150, as appropriate. In additional, a laser and filter arrangement
152 and 154, detector and filter arrangement 156 and 158, and
detector and filter arrangement 160 and 162, can be employed to
monitor the manner in which the droplets are being deflected.
Further details of the charging, deflection, and monitoring of the
droplets are described in copending U.S. patent application Ser.
No. 09/346,692, referenced above.
[0050] Further details of the emission block 140 will now be
discussed with reference to FIGS. 3-10. As illustrated, emission
block 140 includes a support ring 142 which can be made from
stainless steel or any other suitable material. As shown, in
particular, in FIGS. 4-6, support ring 142 has inner groves 144 in
its inner surface and outer groves 146 in its outer surface. A
first flex circuit 148 is mountable in support ring 142.
Specifically, the first flex circuit 148 includes projections 150
that are received into inner groves 144 of support ring 142 to thus
mount the first flex circuit 148 inside support ring 142. As can be
appreciated by one skilled in the art, first flex circuit 148 is an
integrated circuit board arrangement that includes a plurality of
integrated circuits (not shown) and contact pads 152 that have
contacts 154 which are adapted to provide connections to the
circuitry in the first flex circuit 148.
[0051] As further illustrated, a second flex circuit 156 is
mountable to the support ring 142. That is, the second flex circuit
156 includes projections 158 that can be received in the outer
groves 146 of the support ring 142 to thus mount the second flex
circuit 156 to the exterior of support ring 142. An adhesive can be
used to secure the first flex circuit 148 and the second flex
circuit 156 to the support ring 142. Like first flex circuit 148,
second flex circuit 156 is also an integrated circuit arrangement
that includes integrated circuits 160 that are capable of carrying
out certain data processing operation as discussed in more detail
below. The second flex circuit 156 further includes contact pads
162 that include contacts 164 which provide connections to the
circuitry in the second flex circuit 156.
[0052] As further illustrated, the emission block 140, first flex
circuit 148 and second flex circuit 156 are housed within an outer
housing 166 and inner housing 168. As illustrated, the combination
of the support ring 142, first flex circuit 148, second 156, outer
housing 166 and inner housing 168 form openings 170 and 172 as
illustrated in FIG. 7. Each of the openings 170 is configured to
receive a mirror assembly 174 which includes a dichroic mirror 176,
the purpose of which is described in more detail below.
Furthermore, each opening 172 is configured to receive a filter
assembly 180, the purpose of which is described in more detail
below. In this example, emission block 140 is capable of receiving
six mirror assemblies 174-1 through 174-6 and seven filter
assemblies 180-1 through 180-7 (see FIGS. 7 and 10). However, the
emission block 140 can be configured to include any suitable number
of mirror assemblies 174 and filter assemblies 180.
[0053] An example of a mirror assembly 174 is shown in FIG. 8. As
stated above, each mirror assembly 174 includes a dichroic mirror
176 that is capable of passing light having a particular wavelength
(e.g., blue light) while reflecting light of all other wavelengths.
The diachronic mirror assembly 174 includes a memory, such as an
electrically, erasable read-only memory (EEPROM), in which is
stored information pertaining to the type of dichroic mirror 176 in
the mirror assembly 174, along with other information such as the
company of manufacture, the date and place of manufacture and so
on, for purposes described in more detail below. The mirror
assembly 174 further includes contacts 178 that provide electrical
connection with the memory embedded in the mirror assembly 174.
Accordingly, when the mirror assembly 174 is inserted into an
opening 170 as shown, for example, in FIG. 3, the contacts 178 of
mirror assembly 174 engage with the contact 154 on the contact pads
152 of the first flex circuit 148. Accordingly, the circuitry in
the first flex circuit 148 can thus access the information stored
in the memory of the mirror assembly 174 for the purposes described
in more detail below.
[0054] A filter assembly 180 is shown in more detail in FIG. 9.
Filter assembly 180 includes a filter 182 that is capable of
passing light of a certain wavelength (e.g., blue light) while
blocking light of all other wave lengths. Furthermore, like mirror
assembly 174, further assembly 180 includes a memory, such as ROM,
in which is stored information pertaining to the type of filter 182
in the filter assembly 180, the date, place, and company of
manufacture, and so on. Further assembly 180 also includes contacts
184 which provide electrical contact to the memory embedded in the
filter assembly 180. Accordingly, when the filter assembly 180 is
inserted into an opening 172 as shown, for example, in FIG. 3, the
contacts 184 of the filter assembly 180 engage with the contacts
164 on a contact pad 162 of the second flex circuit 156. Hence, the
circuitry in the second flex circuit 156 can then access the
information stored in the memory of the filter assembly 180 for
reasons discussed below.
[0055] As further shown in FIG. 3, for example, emission block 140
include a plurality of detectors 186 which, in this example, are
photomultiplier tubes (PMTs). Each photomultiplier tube detector
186 has an opening therein (not shown) which is aligned with a
dichroic mirror 176 in its respective mirror assembly 174, and with
a filter 182 in its respective filter assembly 180, so that the
detector 186 will receive light passing through its respective
dichroic mirror 176 and filter 182. Each detector 186 further
includes a circuit board assembly 188 that include circuitry for
processing the light received by its respective PMT detector 186,
as well as power and control circuitry for the PMT, as discussed in
more detail below.
[0056] As shown in FIG. 3, for example, and in more detail in FIG.
10, the mirror assemblies 174 are angled so that the side-scatter
laser light entering the emission block 140 from fiber optic cable
130 is reflected to all of the mirror assemblies 174 and to all of
the filter assemblies 180. Specifically, when the laser light
enters the emission block 140 from fiber optic cable 130, the laser
light propagates to mirror assembly 174-1. The dichroic mirror of
mirror assembly 174-1 allows light having a certain wavelength to
pass to filter assembly 180-1, which also allows light of that
wavelength to be detected by its respective detector 186-1.
Detector 186-1 outputs a signal representative of the detected
light, which is processed as described in more detail below.
[0057] As further illustrated, the portion of the laser light
reflected by mirror assembly 174-1 propagates to mirror assembly
174-2, which functions in a manner similar to mirror assembly 174.
That is, the dichroic mirror of mirror assembly 174-2 allows light
of a certain wavelength (e.g., green light) to pass to filter
assembly 180-2 while reflecting light of all other wavelengths.
Accordingly, the light passing to filter assembly 180-2 will pass
through the filter of filter assembly 180-2 and be received by
detector 186-2, while the reflected light will propagate to mirror
assembly 174-3. As can be appreciated from the above description,
mirror assemblies 174-3 through 174-6 will each allow light within
a certain respective wavelength range to pass through to the
corresponding filter assemblies 180-3 through 180-6, respectively,
while reflecting light of all remaining wavelengths. It is noted
that the light reflected by mirror assembly 174-6 will propagate
directly into filter assembly 180-7, because no further reflection
is necessary. Filter assembly 180-7 will therefore allow light
within a respective wavelength to pass to its corresponding
detector 186-7.
[0058] As discussed above, each laser 122 (see FIG. 1) of the flow
cytometer 100 is associated with a respective fiber optic cable 130
and emission block 140. Accordingly, as discussed in more detail
below, if flow cytometer 100 includes, for example, four different
lasers 122, then the flow cytometer will also include four emission
blocks 140, with each emission block 140 being associated with a
respective laser 122 to receive side-scatter laser light in the
manner described above.
[0059] An example of the electronics included in the flow cytometer
100 is shown in block diagram format in FIG. 11. As discussed
above, the flow cytometer 100 includes a controller 118 which, in
this example, includes a data acquisition unit 190, a status and
control unit 192, a droplet control module 222 and a fluidics
control module 224. As indicated, the data acquisition unit 190
includes a processor 194 which, in this example, is a real-time or
near real-time CPU, such as a Pentium III processor or any other
suitable processor. The processor 194 is coupled to the screen LCD
196 of the flow cytometer 100, as well as a sample loader 198 and
sample output device 200. The processor 194 is further coupled to a
hub 202 which provides data to and from work station 204 and
processor 194 as described in more detail below. It is noted that
the processor 194 provides the data pertaining to the event
readings to the work station 204 in packet format in real-time or
near real-time. The hub 202 further provides data to and from
processor 194 and a prepper unit 206 which can be, for example, any
type of sample preparation unit such as that described m U.S.
patent application Ser. No. 09/447,804, referenced above.
[0060] The data acquisition unit 190 further include a plurality of
data acquisition modules 208 that are each capable of acquiring
data from respective circuit board assemblies 188 of the detectors
186 discussed above as described in more detail below. The data
acquisition unit 190 further includes a master data acquisition
module 210 that gathers the data from all of the other data
acquisition modules 208 via a plurality of link-ports 211 and
provides the data to processor 194 as discussed in more detail
below.
[0061] As further illustrated, the processor 194 of data
acquisition unit 190 communicates with the controller 212 of status
and control unit 192 to control, for example, the fluid flow, drop
delay, PMT driving voltage, and so on as described in more detail
below. The status and control unit 192 include PMT modules 214
which, under the control of controller 212, control the driving
voltage of the PMT detectors 186 as discussed in more detail below.
The status and control unit 192 further include a laser control
module 216 which, under control of controller 212, controls
operation of laser 122. The status and control unit 192 also
includes a power and temperature control module 218 that controls,
for example, the power to components of the flow cytometer 100, as
well as the temperature of the sheath and sample fluid.
[0062] In addition, status and control unit 192 further includes an
emission identification (ID) module 220 that receives information
from the first flex circuit 148 and second flex circuit 156
indicative of the locations of the mirror assemblies 174 and filter
assemblies 180. in the emission block 140. That is, as discussed
above, each mirror assembly 174 and filter assembly 180 includes a
memory in which is stored information pertaining to its respective
mirror or filter. The circuitry in the first flex circuit 148 is
capable of accessing the memory in the filter assemblies 180, and
providing the content of this memory to the emission ID module 220.
Likewise, the circuitry in the second flex circuit 156 is capable
of accessing the memories in the filter assemblies 180 and
providing that information to the emission ID module 220. The
emission ID module 220 then can determine whether each of the
mirror assemblies 174 and filter assemblies 180 are in the
appropriate positions based on information pertaining to a desired
configuration stored in a memory that was provided, for example, by
work station 204. If the emission ID module 220 determines that a
mirror assembly 174 or filter assembly 180 is missing or in an
incorrect location in the emission block 140, or if an erroneous or
faulty mirror assembly 174 or filter assembly 180 has been
installed in the emission block 140, emission ID module 220 will
provide the appropriate data to, for example, the controller 212,
which can then provide the data to the processor 194. The processor
194 can then provide this data to, for example, work station 204,
which can display an appropriate error message. This error message
can indicate the location of the incorrect mirror or filter
assembly in the emission block 140, and the work station 204 can
also display the filter and mirror configuration, which therefore
greatly simplifies troubleshooting.
[0063] As further shown in FIG. 11, the master data acquisition
module 210, which is described in more detail below, receives from
the data acquisition modules 208 event data that has been provided
to the data acquisition modules 208 from the PMT detectors 186 of
the emission blocks 140. Prior to running the flow cytometer 100 to
detect events, the work station 204 can download data via the hub
202 and processor 194 to the master data acquisition module 210.
This downloaded data is stored in a memory in the master data
acquisition module 210 and indicates to the master data acquisition
module 210 the channel configuration of the data acquisition
modules 208, so that the master data acquisition module 210 can
recognize which channels of the data acquisition modules 208 are
active, and the type of data (e.g., representative of side scatter
blue light, side scatter red light and so on) that the data from
each channel represents, as discussed in more detail below.
[0064] The master data acquisition module 210 further provides and
receives data to and from the droplet control module 222 and the
fluidics control module 224 to control the operation of the flow
cytometer 100 in the manner described above. For example, the
master data acquisition module 210 can receive high-speed clock
data from the droplet control module 222 that gives the master data
acquisition module 210 a time reference as to the rate of drop
formation (e.g., 50 thousand drops per second). Master data
acquisition module 210 can use this time base to synchronize a
direction command signal which can be, for example, a four bit
binary code, that the master data acquisition module 210 sends to
the droplet control module 222 so that the droplet control module
222 can control the charging unit 147 (see FIG. 2) as appropriate
to achieve the desired charging of the appropriate droplets
containing a cell or particle of interest. By charging the droplet
with the appropriate charge, the droplet control module 222 thus
controls the amount and direction of deflection that the deflection
plates 142 and 144 (see FIG. 2) deflect the charged droplet. The
deflection plates 142 and 144 are included among the sorting
hardware 235 shown in FIG. 11. The droplet can be deflected, for
example, to be received in one of any suitable number (e.g.,
sixteen) collection vessels 142, 146 and 150.
[0065] In addition, the master data acquisition module 210 can
receive data from the processor 194 that has been acquired by, for
example, detectors 120, 156 and 160 that provide information
concerning the status of the break-off point 112 (see FIG. 1) as
well as information pertaining to the droplet sorting. Based on
this data, the master data acquisition module 210 can provide
control signal to the droplet control module 222 to control, for
example, drop delay, droplet formation and so on as discussed above
with regard to FIGS. 1 and 2. processor 194 can further control the
droplet control module 222 to control, for example, a cooling
module 234 and an aerosol management module 236 to control the
temperature of the sorted sample, for example, as well as to
control sorting and aerosol containment management and safety
devices in the flow cytometer 100. It is also noted that the
fluidics control module 224 can control the valve and pump drivers
226, the agitation module 228, the temperature control module 230
and the multiport valve HPLC 232 to control the temperature of the
fluid sample and sheath fluids, to agitate the sample in the sample
reservoir 106 (see FIG. 1), and to control the flow of fluids in
the flow cytometer 100.
[0066] It is further noted that the flow cytometer 100 need not
include all of the electronics shown in FIG. 11. For example, if
the flow cytometer 100 is not equipped to perform droplet sorting,
certain components shown in FIG. 11 can be omitted. As shown in
FIG. 12, the hardware of the data acquisition unit 190 and status
and control unit 192 can consolidated into a data acquisition unit
190-1. The components of the data acquisition unit 190-1, such as
the processor 194, data acquisition modules 204 and master data
acquisition module 210 operate in a manner similar to those
described above with regard to FIG. 11. However, the data
acquisition module 190-1 includes an SCI controller 238 which
performs the operations performed by status and control I/F unit
192 shown in FIG. 11, such as controlling the driving voltages of
the lasers 122 and power and temperature sensor module 218 which
operates as described above. The SCI controller 238 further
controls operation of the driving voltage of detectors 186 in a
manner described below, and receives and processes the mirror and
filter assembly position information received from the emission
block 140 in a manner similar to the emission ID module 220
described above.
[0067] The operation of the above components in relation to the
operation of flow cytometer 100 will now be described. As discussed
above, flow cytometer 100 will typically employ more than one laser
122 to sample more than one type of cell or particle of interest,
or more than one characteristic of a cell or particle of interest.
The following discussion will assume that the flow cytometer 100
includes four lasers 122, each emitting light having a different
wavelength.
[0068] As discussed above and as shown conceptually in FIGS. 13-16,
if the flow cytometer 100 includes four lasers 122-1 through 122-4,
then the flow cytometer 100 will include four corresponding fiber
optic cables 130-1 through 130-4 that feed the respective
side-scatter laser lights to the respective emission blocks 140-1
through 140-4. As further shown, the laser light emitted from these
respective lasers 122-1 through 122-4 strike respective
interrogation points 124-1 through 124-4 on the fluid stream. In
this example, the interrogation points are displaced by about 133
micrometers along the direction of flow of the fluid stream, which
translates into a spacing of about 22 microseconds for a fluid
stream flowing at a rate of 6 meters per second. As shown in FIG.
16, this spacing also permits inter-laser mixing to occur. For
example, the side scatter laser light from interrogation point
124-3 can enter the fiber optic cable 130-4 dedicated to receive
side scatter laser light from interrogation point 124-4. The mirror
assemblies 174 and filter assemblies 180 in the emission blocks
140-1 through 140-4 can be configured to eliminate any light of
undesired wavelengths as discussed above, in the event that
unwanted inter-laser mixing occurs.
[0069] Further details of the relationship between the detectors
186, a data acquisition module 208, master data acquisition module
210, processor 194 (real time CPU) and the work station will now be
described with regard to FIGS. 17-21. In this arrangement, each
data acquisition module 208 can receive data from four detectors
186 from any of the emission blocks 140. For purposes of this
discussion, data acquisition module 208 is configured to receive
side scatter laser light that has been generated by the four
different wavelength lasers 122-1 through 122-4.
[0070] As illustrated, the analog data signals from the detectors
186 are input to their respective data acquisition module 208 as 2
MHz bandwidth (BW) analog signals. Further details of the data
acquisition module are shown in FIGS. 18 and 19. That is, the
signal from each detector 186 is input to a respective
analog-to-digital (A/D) converter 240 where the analog data is
converted into digital data. As illustrated, each A/D converter 240
have differential inputs to maximize common mode rejection of the
received analog signals. The frequency (e.g., 10 MHz) at which the
A/D converters 240 are operating enable the A/D converters 240 to
take multiple samples (e.g., 10 or 20, or more) of the waveform as
shown in FIG. 21 in real-time or near real-time. As indicated, the
intensity of the signal will typically increase to a maximum when
the particle or cell of interest is at the center of the
interrogation point, and then drop-off to a minimum as the cell
passes out of the interrogation point 124. Accordingly, each
individual sample of the waveform will have a value representing
the characteristic (e.g., height) of that sampled portion of the
waveform. This sampling of the entire or substantially the entire
waveform improves the details at which the waveforms can be
analyzed and compared, for example, to other waveforms
representative of other events. Accordingly, this sampling allows
for a more detailed sampling of the characteristics of each
event.
[0071] The digital data output by each A/D converter 240 is
provided to a respective delay circuit 244 which imposes a
respective delay on the digital data as described in more detail
below. As shown, for example, in FIG. 19, the delay imposed by each
delay circuit 244 is set to compensate for the delays between the
interrogation points 124-1 and 124-4 as shown in FIG. 15 or, in
other words, to compensate for the time delay that occurs between
when the side scatter light representative of a particle or cell of
interest at interrogation point 124-1 is received by a detector 186
in emission block 140-1 and when the side scatter light
representative of that particle or cell of interest reaching
interrogation points 124-2 through 124-4 are subsequently received
by detectors 186 in their respective emission blocks 140-2 through
140-4.
[0072] The digital data from each delay circuit 244 is provided to
a respective channel field programmable gate array (FPGA) circuit
246, which provide the data to a Super Harvard Architecture
Computer (SHARC) unit 248. It is noted that each channel FPGA
circuit 246 can process the characteristics of the data samples to
produce data representing a single characteristic of the analog
waveform, such as the width or height of the waveform, if desired,
instead of passing all of the samples (e.g., 20 samples per
waveform as discussed above) to the SHARC unit 248. Also, the
channel FPGA circuits 246 will add a time stamp to their respective
data prior to passing the data to the SHARC 248. Under the control
of a programmable logic device, versa-module Eurocard interface
(PLD VME I/F) unit 250 and a trigger FPGA unit 252, the SHARC unit
248 provides the digital data via a link port 211 to the master
data acquisition module 210 as indicated.
[0073] Specifically, prior to running the flow cytometer 100 to
detect the events, the workstation 204 can download channel data to
the trigger FPGA unit 252 of each data acquisition module 208 via
the hub 202 and processor 194. This channel data indicates to the
channel FPGA circuits 246 whether they should collect the data from
their respective delay circuits 244, that is, whether they are
receiving data on an active channel. The channel data further
indicates to the trigger FPGA unit 252 when the trigger FPGA unit
252 should trigger the SHARC 248 to transfer the event data
received in parallel from the channel FPGAs 246 to the master data
acquisition module 210 via the link port 211.
[0074] Details of the master data acquisition data module 210 are
shown FIG. 20. That is, the master data acquisition module 210
includes a multi-SHARC unit 256 that includes a SHARC event
classification unit 258, a SHARC drop classification unit 260 and a
SHARC event assembly unit 262, the details of which are described
below. The master data acquisition module 210 further include a
gate FPGA 264, a logarithmic look-up table 266, and a data FIFO
unit 268. Furthermore, the master data acquisition data module 210
includes an FPGA module 270 that includes a drop control FPGA 272
and a trigger FPGA 274. The master data acquisition module further
include a PLD VME I/F 276. The details of these components are
described below.
[0075] Specifically, prior to running the flow cytometer 100 to
detect the events, the workstation 204 can download channel data to
the trigger FPGA unit 274 of master data acquisition module 210 via
the hub 202 and processor 194. This channel data indicates to the
trigger FPGA units 252 of each data acquisition module 208 whether
they should trigger their respective SHARC 248 to transfer the
event data received in parallel from the channel FPGAs 246 to the
master data acquisition module 210 via their respective link port
211. That is, when the trigger FPGA units 252 provide their
respective indications to the trigger FPGA unit 274 indicating that
event data has been received on their appropriate respective
channels, the trigger FPGA unit 274 will signal the trigger FPGA
units 252 to trigger their respective SHARCs 248 to transfer the
event data received in parallel from the channel FPGAs 246 to the
master data acquisition module 210 via the link port 211.
[0076] When the master data acquisition module 210 receives the
event data via the linkports 211, the event data is input to the
SHARC event assembly 262. The SHARC event assembly 262 assembles
the data into lists, tables or buffers based on their time-stamp
that has been added by the channel FPGAs 246. That is, the SHARC
event assembly 262 uses the time stamps to determine which data is
associated with which event.
[0077] If no sorting of cells is to be performed, the SHARC event
assembly 262 passes the lists, tables or buffers of data to the
data FIFO unit 268. The data FIFO unit 268 sends the lists, tables
or buffers of the data via the VME bus 254 to the processor 194.
The processor 194 can then provide the data to the work station 204
for further display in, for example, a scatter plot diagram, a
graphical representation, and so on.
[0078] However, if cell sorting is to be performed, data received
by the SHARC event assembly unit 262 is processed by the SHARC
event classification unit 258 and SHARC drop classification unit
260. For example, the flow cytometer 100 can be run to sample a
portion of the cell sample to therefore provide initial sample data
to the work station 204 as discussed above. The work station 204
can display the detected events on, for example, a scatter plot
which can be reviewed by the operator. The operator can select
certain cells of interest to be sorted by selecting, for example, a
region on an interactive display screen of the work station 204.
The work station 204 can then pass the desired cell sorting data to
the master data acquisition module 210 via hub 202 and processor
194. The master data acquisition module 210 stores this cell
sorting data in, for example, the logarithmic lookup SRAM 266.
[0079] When the operator reactivates the flow cytometer 100 to
continue processing the sample, the SHARC event classification unit
258 and SHARC drop classification unit 260 can access the data in
the logarithmic lookup SRAM 266 in real-time or near real-time to
determine which data received by the SHARC event assembly unit 262
represents cells to be sorted. The SHARC event classification unit
258 and SHARC drop classification unit 260 can then provide signals
to the Drop Control FPGA 272 which can provide the appropriate
direction command signal to the droplet control module 222 so that
the droplet control module 222 can control sorting as discussed
above. The SHARC event assembly 262 can then pass the lists, tables
or buffers of data to the data FIFO unit 268, which sends the
lists, tables or buffers of the data via the VME bus 254 to the
processor 194 as discussed above. The processor 194 can then
provide the data to the work station 204 in real-time or near
real-time for further display in, for example, a scatter plot
diagram, a graphical representation, and so on.
[0080] Additionally, the event data can be used to process the
sample waveforms in various ways. For example, the above system, in
particular, the controller 212 (FIG. 11) or SCI controller 238
(FIG. 12) can adjust system can adjust the voltages applied to the
detector 186 (PMTs) to adjust the relative zero point of the PMT
detector 186. For example, as shown in FIG. 22, the PMT and circuit
board 188 includes a DC high voltage power supply 280 that provide
the driving voltage to the PMT socket 282 that drives the PMT. The
current from the PMT generated upon, for example, detection of side
scatter light as described above is converted by a current voltage
converter 284 so that the voltage signal is provided to the
respective channel data acquisition module 208 as described above.
Voltage control and serial control signal are provided from the PMT
controllers 214 in, for example, the respective channel data
acquisition module 208 to adjust the base voltage of the PMT, to
therefore adjust the relative zero point of the PMT. --Accordingly,
this adjustment can be used to perform the gain adjustment as
shown, for example, in FIG. 23 to increase the height of the
smaller waveform to be consistent with the heights of the red and
blue waveforms.
[0081] In addition, as shown in FIGS. 24-27, the SHARC event
assembly 262 the master data acquisition module 210 can compare the
entire sample wave form of data obtained from different detectors
186 and can perform different types of processing functions on this
data in a real time or near-real time basis. For example, the event
data representative of the red side scatter light signal received
at time T can be delayed so that it can be compared with the event
data representative of the blue side scatter light signal received
at time T+1 as shown in FIG. 24, so that the signals can be time
correlated as shown in FIG. 25. Furthermore, as shown in FIGS. 26
and 27, the data signals can be processed to remove crosstalk that
can occur as discussed above. In this event, the blue data
represented as the "blue+red crosstalk" can be processed to remove
a percentage of the red signal that is affecting the magnitude of
the blue data, so that the magnitudes of the blue and red data can
be made similar for comparison as shown in FIG. 27.
[0082] Although only a few exemplary embodiments of the present
invention have been described in detail above, those skilled in the
art will readily appreciate that many modifications are possible in
the exemplary embodiments without materially departing from the
novel teachings and advantages of this invention. Accordingly, all
such modifications are intended to be included within the scope of
this invention as defined in the following claims.
* * * * *