A data collection method for scanning a scan window comprising one or more channels is described. In the method of the invention an integrated signal (S) is measured across a scan window including one or more channels using an integrating detector. Next, a velocity-normalized integrated signal (Sn) is determined based on the integrated signal (S) and a scan velocity.
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1. A data collection method for scanning a scan window comprising one or more channels comprising the steps of:
detecting an integrated signal (S) across a the scan window comprising one or more channels using an integrating detector; and
calculating a velocity-normalized integrated signal (Sn) as a function of a scan velocity and the integrated signal S.
0. 38. A data collection method for scanning a scan window comprising:
detecting an integrated signal (S) across the scan window comprising one or more channels using an integrating detector;
determining an integration time (ti) for the integrated signal; and
calculating a velocity-normalized integrated signal (Sn), the calculating comprising dividing the integrated signal (S) by the integration time (ti).
0. 36. An apparatus for scanning one or more channels comprising:
means for detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector; and
computer means for receiving the integrated signal S and determining a scan velocity and for calculating a velocity-normalized integrated signal (Sn) as a function of the scan velocity and the integrated signal S.
16. An apparatus for scanning a plurality of channels comprising:
means for detecting an integrated signal (S) across a scan window comprising one or more the plurality of channels using an integrating detector; and
computer means for receiving the integrated signal S and determining a scan velocity and for calculating a velocity-normalized integrated signal (Sn) as a function of the scan velocity and the integrated signal S.
21. A program storage device readable by a machine, tangibly embodying a program of instructions executable by a machine to perform method steps to scan a scan window comprising one or more channels, said method steps comprising:
detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector; and
calculating a velocity-normalized integrated signal (Sn) as a function of a scan velocity and the integrated signal S.
17. An apparatus for scanning a scan window having one or more channels comprising:
an integrating detector;
a scanner for effecting a scanning of the integrating detector relative to a the scan window comprising one or more channels, wherein an integrated signal (S) is detected by scanning the integrating detector relative to the scan window; and
a computer for receiving the integrated signal S and for determining a scan velocity and for calculating a velocity-normalized integrated signal (Sn).
0. 52. An apparatus for scanning one or more channels comprising:
means for detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector;
means for determining an integration time (ti) for the integrated signal; and
computer means for receiving the integrated signal (S) and the integration time (ti), and for determining a velocity-normalized integrated signal (Sn), the determining comprising dividing the integrated signal (S) by the integration time (ti).
0. 62. A program storage device readable by a machine, tangibly embodying a program of instructions executable by a machine to perform a method to scan a scan window comprising one or more channels, said method comprising:
detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector;
determining an integration time (ti) for the integrated signal (S); and
calculating a velocity-normalized integrated signal (Sn), the calculating comprising dividing the integrated signal (S) by the integration time (ti).
0. 56. An apparatus for scanning a scan window having one or more channels comprising:
an integrating detector;
a scanner configured to scan the integrating detector relative to the scan window, wherein an integrated signal (S) is detected by scanning the integrating detector relative to the scan window;
a timer configured to determine an integration time (ti) for the integrated signal; and
a computer configured to receive the integrated signal (S) and the integration time (ti), and to determine a velocity-normalized integrated signal (Sn), the determining comprising dividing the integrated signal (S) by the integration time (ti).
2. The method of
determining a scan velocity, v; and
dividing the integrated signal S by the scan velocity v.
3. The method of
measuring a channel width (w);
determining a time for traversing the channel width (t); and
computing a the velocity-normalized integrated signal according to the equation Sn=S/(w/t).
4. The method of
9. The method of
10. The method of
11. The method of
12. The method of
14. The method of
22. The program storage device of
determining a scan velocity, v; and
dividing the integrated signal S by the scan velocity v.
23. The program storage device of
measuring a channel width (w);
determining a time for traversing the channel width (t); and
computing a the velocity-normalized integrated signal according to the equation Sn=S/(w/t).
24. The program storage device of
25. The program storage device of
0. 26. The method of
wherein the calculating the velocity-normalized integrated signal comprises dividing the integrated signal (S) by the integration time (ti),
and wherein the scan window comprises more than one channel.
0. 27. The method of
0. 28. The method of
0. 29. The apparatus of
0. 30. The apparatus of
0. 31. The apparatus of
0. 32. The apparatus of
0. 33. The apparatus of
0. 34. The program storage device of
0. 35. The program storage device of
0. 37. The apparatus according to
0. 39. The method of
0. 40. The method of
0. 41. The method of
0. 42. The method of
0. 43. The method of
0. 44. The method of
0. 45. The method of
0. 46. The method of
0. 47. The method of
0. 48. The method of
0. 49. The method of
0. 50. The method of
0. 51. The method of
0. 53. The apparatus of
0. 54. The apparatus of
0. 55. The apparatus of
0. 57. The apparatus of
0. 58. The apparatus of
0. 59. The apparatus of
0. 60. The apparatus of
0. 61. The apparatus of
0. 63. The program storage device of
0. 64. The program storage device of
0. 65. The program storage device of
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This invention relates to methods, software and apparatus useful for scanning one or more channels using an integrating detector. More specifically, this invention relates to means for scanning which compensates for variable scanning velocities.
Scanning refers to a process whereby an integrated signal is obtained from one or more channels using an integrating detector which serially interrogates each channel. Such scanning systems are used in a variety of applications including text scanners, bar-code scanners, and electrophoresis scanners. A particularly important class of scanning systems are utilized in automated fluorescence-based DNA sequencing systems, e.g., U.S. Pat. Nos. 4,811,218; 5,091,652, 5,274,240, 5,102,785 and 5,543,026.
There are two important classes of scanning systems: position-based scanners and time-based scanners. In time-based scanners, a fixed integration time is used to collect an integrated signal from one or more channels of an object to be scanned. A feature of time-based scanning systems is that they provide low levels of time-dependent background signal. However, time-based scanners have the drawback that they generally display poor position repeatability, largely because of non-uniform scanning velocities due to acceleration/deceleration of the scanner and/or imperfect scanner repeatability. That is, the location of scan channels can vary from scan to scan. For example, in the case of an electrophoresis scanner, poor position repeatability may lead to poor lane tracking performance, i.e., it becomes impossible to distinguish a lane from neighboring lanes. This problem can become particularly severe when the density of lanes becomes high.
In position-based scanners, the integration time is based on a width of a channel and a scan velocity. Thus, rather than integrating a signal over a specified time, the signal is integrated over a specified distance, i.e., a channel width. Position-based scanners generally have superior positional repeatability. Thus, in the electrophoresis scanning application, position-based scanners exhibit superior lane tracking performance. However, position-based scanners display a high level of background noise because of non-uniform integration times resulting from the non-uniform scanning velocities mentioned above. Because signal strength is proportional to integration time, such non-uniform integration times result in high levels of time-dependent background noise.
Thus, it would be desirable to produce a scanner which combines the superior position repeatability of a position-based scanner with the low noise level of a time-based scanner.
The present invention is directed towards the discovery of scanning systems which normalize an integrated signal intensity with respect to a scan velocity in order to achieve superior scanning performance.
It is an object of the present invention to provide a scanning system which provides superior positional repeatability.
It is another object of the present invention to provide a scanning system which has a reduced sensitivity to non-uniform scanning velocity.
In a first aspect, the foregoing and other objects of the invention are achieved by a method for scanning a scan window comprising one or more channels comprising the steps of first detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector, then calculating a velocity-normalized integrated signal (Sn).
In another aspect, the present invention comprises a program storage device readable by a machine, tangibly embodying a program of instructions executable by a machine to perform the above method steps.
In yet another aspect, the present invention includes An apparatus for scanning a plurality of channels comprising means for detecting an integrated signal (S) across a scan window comprising one or more channels using an integrating detector, and computer means for calculating a velocity-normalized integrated signal (Sn).
These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.
FIG. 1 is a process flow diagram illustrating the steps of the scanning method of the invention.
FIG. 2 is a process flow diagram illustrating the steps of the velocity-normalization aspect of the scanning method of the present invention.
FIG. 3 is a plot of average signal strength versus channel position for 78 scans of a 480-channel scan window using position-based data collection without velocity normalization.
FIG. 4 is a plot similar to FIG. 3 but showing collected employing the velocity-normalized position based collection method of the present invention.
FIG. 5 is a plot of signal-to-noise ratio versus channel number for data collected across a 480-channel scan window without the velocity normalization.
FIG. 6 is a plot similar to FIG. 5 but showing data collected employing velocity-normalization.
FIG. 7 is a plot of signal-to-noise ratio versus channel position across a 388-channel scan window using conventional time-based data collection.
FIG. 8 is a plot similar to FIG. 7 but showing data collected employing velocity-normalization.
An exemplary confocal fluorescence detection system for use with capillary arrays is shown in FIG. 9. An argon ion laser (Model 2020, Spectra-Physics, Mountain View, Calif.), not shown, is used as the excitation source. The laser beam is expanded to 5 mm diameter, collimated, and then directed through a 32 X, N.A. 0.4 infinite conjugate objective 11(LD Plan-Achromat 440850, Carl Zeiss, West Germany) by a long-pass dichroic beamsplitter 12(480 DM, Omega Optical, Brattleboro, Vt.). The dichroic beam splitter 12 reflects the excitation laser beam into the objective 11 but transmits fluorescent light collected by the objective which is Stokes shifted to longer wavelengths. The objective focuses the exciting laser on the sample and gathers the fluorescence with very high collection efficiency. The use of an infinite conjugate objective permits vertical adjustment of the probe volume by translating the objective with the mount 13 secured to the base 14 with no significant perturbation of the optical alignment. The focused 1 mW, 488 nm wavelength beam is focused to a 10 μm beam diameter and a 25 μm confocal beam parameter. The fluorescence emission is passed back through the long-pass dichroic beam splitter 12 mounted on the base 14 to reduce laser interference and to separate the excitation and detection paths. The fluorescence is then focused by a 75 mm focal length lens 16 mounted on the base 14 onto a 400 μm pinhole which serves as the confocal spatial filter. The light passing through the pinhole is filtered by a 488 nm rejection band filter (488 RB filter, Omega Optical, Brattleboro, Vt.), a long-pass cutoff filter (Schott GG-495, Esco, Oakridge, N.J.), a bandpass fluorescence filter (530 DF60, Omega Optical, Brattleboro, Vt.), all mounted within the housing 17, followed by detection with a cooled photomultiplier tube 18(RCA 31034A, Burle Industries, Lancaster, Pa.). The spatial filter, the optical filters and photomultiplier tube are mounted on base 14. The output of the phototube is amplified and filtered with a low-noise amplifier (SR560, Standford Research Systems, Sunnyvale, Calif.), digitized with a 12 bit analog-to-digital board (DASH-16 F, metra-Byte, Taunton, Mass.) and stored in an IBM PS/2 microcomputer. The capillary array comprises a plurality of capillaries 21 having their ends 22, 23 extending into wells 24, 26 between which a high voltage is applied for electrophoresis. The ends 22 may be separated for individual manipulation and loading. A portion 27 of the capillaries is maintained in side-by-side parallel coplanar relationship by a holder 28, FIG. 10. The holder 28 includes a window 32 through which the beam can be focused on the interior volume of the capillaries. The holder 28 is mounted on a translation stage 30 (Model 4000, Design Components, Franklin, Mass.), which is actuated by stepper motor 31(see FIG. 9).
The following is a list of parameters used for scaling the first few channels. The information recorded is
<Ch = 0> <tic = FA2F>
<Ch = 1> <tic = 2CEC> <time = 32BD> <0: 0C42 −> 0C7E> <1: 0279 −> 0288> <2:
050D −> 052E> <3: 09B4 −> 09F5>
<Ch = 2> <tic = 60E4> <time = 33F8> <0: 0C3F −> 0C43> <1: 02B2 −> 02B3> <2:
0508 −> 050A> <3: 09FB −> 0A00>
<Ch = 3> <tic = 9567> <time = 3483> <0: 0C7B −> 0C67> <1: 029E −> 0298> <2:
0568 −> 055C> <3: 09FF −> 09EA>
<Ch = 4> <tic = C95D> <time = 33F6> <0: 0BEB −> 0BF0> <1: 02EB −> 02EC> <2:
0517 −> 0519> <3: 09BD −> 09C2>
<Ch = 5> <tic = FD6E> <time = 3411> <0: 0C11 −> 0C11> <1: 02A7 −> 02A7> <2:
050C −> 050C> <3: 09F0 −> 09F0>
<Ch = 6> <tic = 316A> <time = 33FC> <0: 0C07 −> 0C0B> <1: 0279 −> 027A> <2:
052E −> 0530> <3: 0A0D −> 0A11>
<Ch = 7> <tic = 65B6> <time = 344C> <0: 0C36 −> 0C2C> <1: 027F −> 027C> <2:
051F −> 0519> <3: 09BF −> 09B4>
<Ch = 8> <tic = 99BF> <time = 3409> <0: 0C3E −> 0C40> <1: 02AC −> 02AC> <2:
04EC −> 04ED> <3: 098F −> 0991>
<Ch = 9> <tic = CDDA> <time = 341B> <0: 0BEA −> 0BE9> <1: 0277 −> 0276> <2:
0507 −> 0506> <3: 09CF −> 09CD>
<Ch = 10> <tic = 015C> <time = 3382> <0: 0BF5 −> 0C0D> <1: 0283 −> 0289> <2:
051F −> 052D> <3: 09B8 −> 09D3>
<Ch = 11> <tic = 3535> <time = 33D9> <0: 0BCF −> 0BD6> <1: 0267 −> 0269> <2:
0507 −> 050C> <3: 09F2 −> 09FD>
<Ch = 12> <tic = 691E> <time = 33E9> <0: 0BDD −> 0BE4> <1: 029F −> 02A1> <2:
052B −> 052F> <3: 09CD −> 09D5>
<Ch = 13> <tic = 9D91> <time = 3473> <0: 0C3D −> 0C2D> <1: 02AE −> 02A9> <2:
050F −> 0506> <3: 0A0A −> 09F8>
<Ch = 14> <tic = D16C> <time = 33DB> <0: 0BBF −> 0BC8> <1: 0277 −> 0279> <2:
053C −> 0541> <3: 09DC −> 09E6>
<Ch = 15> <tic = 05B5> <time = 3449> <0: 0BC9 −> 0BC0> <1: 0283 −> 0280> <2:
0519 −> 0513> <3: 09DA −> 09D0>
<Ch = 16> <tic = 39CD> <time = 3418> <0: 0C13 −> 0C12> <1: 027D −> 027C> <2:
0542 −> 0541> <3: 09CE −> 09CD>
<Ch = 17> <tic = 6DD9> <time = 340C> <0: 0C17 −> 0C18> <1: 02A4 −> 02A4> <2:
04F8 −> 04F8> <3: 0A23 −> 0A24>
<Ch = 18> <tic = A1DA> <time = 3401> <0: 0BDD −> 0BE0> <1: 0274 −> 0274> <2:
0505 −> 0506> <3: 09DF −> 09E2>
<Ch = 19> <tic = D640> <time = 3466> <0: 0BAF −> 0BA2> <1: 02BE −> 02B9> <2:
04CF −> 04C7> <3: 09CF −> 09BF>
<Ch = 20> <tic = 0955> <time = 3315> <0: 0BE6 −> 0C11> <1: 0253 −> 025E> <2:
050C −> 0524> <3: 09A7 −> 09D7>
<Ch = 21> <tic = 3D9A> <time = 3445> <0: 0B87 −> 0B7F> <1: 029A −> 0297> <2:
053E −> 0539> <3: 09B6 −> 09AD>
<Ch = 22> <tic = 7149> <time = 33AF> <0: 0BC7 −> 0BD7> <1: 0287 −> 028B> <2:
04DF −> 04E8> <3: 09C8 −> 09DB>
<Ch = 23> <tic = A5A2> <time = 3459> <0: 0BF7 −> 0DEB> <1: 02AF −> 02AB> <2:
0515 −> 050E> <3: 0A2B −> 0A1D>
<Ch = 24> <tic = D9C2> <time = 3420> <0: 0BE7 −> 0BE5> <1: 027F −> 027E> <2:
0519 −> 0517> <3: 09CF −> 09CC>
<Ch = 25> <tic = 0DBC> <time = 33FA> <0: 0C0E −> 0C12> <1: 02AA −> 02AB> <2:
0503 −> 0505> <3: 0A12 −> 0A17>
All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although only a few embodiments have been described in detail above, those having ordinary skill in the scanning art will clearly understand that many modifications are possible in the preferred embodiment without departing from the teachings thereof. All such modifications are intended to be encompassed within the following claims.
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