JP4119383B2 - Electrophoresis system and method for correcting an electrophoresis gel image - Google Patents

Description

  The present invention relates to an electrophoresis system for removing non-uniformity of an electrophoresis system and a method for correcting an electrophoresis gel image.

  In the biotechnology field, fluorescent dyes are routinely used as sensitive non-isotopic labels. These labels identify and locate a wide range of cellular structures from malignant tumors to specific chromosomes in the DNA sequence. A wide range of devices have been designed for reading fluorescently labeled samples.

  Gel electrophoresis is one technique that is widely used to identify specific molecules and other labeled units along with fluorescent dyes and other markers. In this technique, an electric field is used to move the labeling unit into a gel or other solution.

  US Pat. No. 4,874,492 discloses a gel electrophoresis system in which a sample is treated with a fluorescent marker before being added to the electrophoresis gel. The gel is illuminated with a UV source and the fluorescence pattern is detected by a cooled charge coupled device (CCD) two-dimensional detector array. The CCD array is cooled to at least −25 ° C. to improve photosensitivity and increase dynamic range.

  U.S. Pat. No. 5,162,654 discloses a system for optically determining which of the four fluorophores are fluorescent in an electrophoresis gel. The fluorescence emitted by the gel first passes through four separate bandpass filters and then through four wedge prisms. With such an optical configuration, the emitted fluorescence is imaged in the four dispersed regions of the detector array. The specific fluorophore excited by the radiation source is determined by comparing the relative intensities of the fluorescence detected in the four detection regions.

  In US Pat. No. 5,294,323, the disclosed gel electrophoresis system utilizes a vertical electrophoresis plate. The laser beam passes horizontally through the gel in a direction perpendicular to the longitudinal axis of the electrophoresis plate. The emitted fluorescence is reflected by the solid state imaging sensor so that the reflection pattern is parallel to the direction of the laser beam.

  U.S. Pat. No. 5,324,401 discloses a fluorescence detection system for capillary electrophoresis that simultaneously excites and detects fluorescent probes within a plurality of capillaries. The excitation source is a laser coupled to a capillary through the optical fiber bundle. Fluorescence from the capillary array is focused through a lens and imaged and analyzed by a CCD camera.

  Published in Analytical Biochemistry 163, 446-457 (1987) “Electronic Imaging System for Direct and Rapid Quantification of Fluorescence from Electrophoretic Gels: Application to Ethidium Bromide Stained DNA” In a Sutherland et al. Paper entitled, the authors describe an imaging system that uses a CCD camera. CCD cameras quantify fluorescence received from electrophoresis gels, chromatograms and other sources. This paper describes several causes of non-uniformity that affect the ability of the system to obtain accurate results.

  From the foregoing, it is apparent that there is a need for an improved electrophoretic device that enables accurate quantitative fluorescence measurements.

  The present invention provides a method and apparatus for correcting non-uniformity of an electrophoretic device. Such non-uniformity arises from the imaging system. By correcting these non-uniformities, quantitative measurement of the electrophoresis gel is possible, and information obtained from the electrophoresis analysis is increased.

  In a form of the invention, the non-uniformity due to the lens assembly is characterized by passing a secondary source of known uniformity through the lens assembly. If desired, the same mirror used to determine source non-uniformity can be used to determine lens non-uniformity. The secondary source is a linear source with either a continuous source or a series of point sources. As described above, the correction file used for normalization of the image file is created and stored.

  In one form of the invention, a platform for holding an electrophoretic gel, a first light source for illuminating the electrophoretic gel, and radiation from a labeled region of the electrophoretic gel is imaged on a first detector. A first lens assembly for measuring and an intensity pattern of a second light source passing through the first lens assembly is measured by the first detector to determine a first lens assembly profile. A mirror for reflecting the illumination from the second light source to the first detector, and the first detector using the first lens assembly profile to normalize the image of the electrophoretic gel. And a processor connected to each other.

  According to the method of correcting an electrophoretic gel image according to the present invention, a part of light from a first light source array is reflected to a detector array through a first lens assembly, and an intensity pattern is generated by the detector array of the first light source array. Measuring and determining a first lens assembly modification file from the measured intensity pattern; storing the first lens assembly modification file; illuminating the electrophoresis gel with an electrophoresis gel illumination source; and at least one of the illuminated gels Two labeled areas fluoresce, the illuminated electrophoretic gel is imaged onto a second detector array, an electrophoretic gel image file is stored, and the electrophoretic gel image file is normalized with the first lens assembly modification file. And each step of storing the normalized electrophoresis gel image file is provided. To.

  The nature and advantages of the present invention may be understood with reference to the remaining portions of the specification and drawings.

  FIG. 1 is a cross-sectional view of a prior art gel electrophoresis apparatus. In this system, the gel plate 101 is illuminated by a light source 103. The light source 103 includes a plurality of individual light bulbs 104. The light from the source 103 causes the fluorophore or other fluorescent material contained in a particular region of the sample 101 to emit fluorescence. The emitted fluorescence passes through one or more lenses 107 and is imaged on the detector 109. Based on the received image, the fluorescent region of the sample 101 can be determined.

  The intensity of fluorescence from sample 101 includes additional information, such as the quantity of fluorescent material, but the ability to quantify this information has been limited so far due to non-uniformity of the illumination source, imaging optics and detectors. It was.

  Detector non-uniformities are most easily removed. For example, a number of CCD arrays are available that provide a linear response over a wide range of intensity levels. In general, these arrays also provide a very uniform response from pixel to pixel, dramatically reducing the non-uniformity resulting from the detector.

  The light intensity from individual bulbs 105 is relatively uniform over most of the bulb length. At both ends of the bulb, the brightness level shows a slight decrease in intensity. This degradation can be minimized by utilizing reflectors, masks, diffusion filters, or combinations thereof. The effect of the decline can also be minimized by simply using a long bulb. By extending the bulb, the bulb end where the brightness level is reduced passes the sampling area of the device so that only a relatively uniform length of the bulb is placed under the sample.

  FIG. 2 is an intensity profile of one bulb 105 measured perpendicular to the bulb axis. As expected, as the distance from the bulb increases, a peak 201 of the profile appears just above the bulb where the intensity drops rapidly. Dip 203 is the result of the scribe mark on the stage. FIG. 3 shows the intensity profile of the source 103 measured perpendicular to the axis of the bulb 105. The peak 301 is on the center line of the individual bulbs 105 and the valley 303 represents the midpoint between the bulbs. Source uniformity is further improved by increasing the number of bulbs and reducing the separation between the bulbs.

  FIG. 4 is an illustration of another method of improving illumination source uniformity. In this method, sample 101 is illuminated by one source 401. Although the source 401 is composed of one light bulb as shown, the source 401 can be composed of a plurality of light bulbs. The source 401 is scanned in a direction 403 perpendicular to the bulb axis to take advantage of the high intensity uniformity along the central bulb portion. The effect of reduced intensity near the bulb end can be minimized by extending the bulb by a distance 405 past the edge of the sample 101. Since the source 401 scans in a direction perpendicular to the bulb uniformity region, the sample 101 is illuminated uniformly as long as the scan speed is constant.

  FIG. 5 is a block diagram outlining the main steps of correcting an image for lens non-uniformity according to the present invention. First, the non-uniformity of the lens assembly is measured (step 501). Many techniques can be used to determine lens non-uniformity, some of which are described below. After characterizing the lens assembly for aperture and magnification for the initial set, these settings are changed (steps 503 and 505) to characterize the lens. This process continues until the lens assembly is characterized for the aperture setting range and the magnification setting range. Then, a lookup table or matrix is created using this information (step 507).

  Once the lens characterization lookup table is created, this data is converted to a modified file lookup table (step 509). This step may be necessary to have a one-to-one correspondence between the number of modified data points per modified file and the number of data points from the sample imaging process. Depending on the technique used to characterize the lens assembly, the number of modified data points may result in significantly less than the number of sample points. In this case, the additional modified data points are filled by the data processor using well-known interpolation techniques, and any lens assembly symmetry is utilized.

  After creating the modified file lookup table, the electrophoresis sample gel is imaged using the system (step 511). At this time, the user can view the uncorrected sample image or store the sample file of the uncorrected image for correction and display it later. Before modifying the sample image, the aperture and magnification settings used to acquire the sample image must first be determined (step 513), but this data is used to find the appropriate modified file in the modified file lookup table. Is necessary. This information can be determined automatically by the system or the user can simply enter the lens settings.

  In order to remove the non-uniformity of the lens assembly from the sample image, the sample file is normalized by dividing by the corresponding correction file (step 515). Prior to normalization, the modified file can first be smoothed using a smoothing function well known in the art. This smoothing function ensures that the noise source of the modified file is not amplified during the normalization process. Finally, the modified sample image is displayed (step 517) or stored (step 519) for future use.

  One way to remove the effects of lens and detector non-uniformity is to use a reference or calibration standard. During calibration, the standard replaces the sample. A preferred calibration standard is a piece of fluorescent glass or plastic that fluoresces uniformly when illuminated by a source. It is desirable for the standard to fluoresce with the same wavelength as the label used with the sample, so that the standard accurately reproduces the lens and detector non-uniformities.

In practice, the user first obtains an image represented by I1 (x, y) of the sample in question. The sample is then replaced with the standard, and the second image, I2 (x, y) is acquired. The second image is known as a flat field image. In order to obtain a corrected image, it is necessary to perform the third exposure with the camera shutter completely closed. The third image, D0 (xy), represents the dark field. The modified image, CI (x, y) is represented by:
CI (x, y) = _{[{I 1 (x, y)} -D 0 (x, y)} / I 2 (x, y) -D 0 (x, y)} ] * M
Where M is equal to the average I2 (x, y).

  While the above technique can be used to obtain an accurate and thus quantitative image of the sample, this technique is impractical for many applications given a uniform illumination source. This technique requires the user to change the sample and perform a series of measurements to obtain a single modified image.

  FIG. 6 is a diagram of another embodiment of the present invention. As in the prior art system shown in FIG. 1, the fluorescence from the sample 101 is imaged onto the detector 109 by the lens 107. In this example, the calibration standard 601 is moved on stage 603 if the user wishes to obtain a modified image. In an embodiment of the present invention, standard 601 is placed in close proximity to the entrance aperture of lens 107. The stage 603 is preferably a rotary stage. As in the previous technique, three images are required: a sample image, a calibration image, and a dark field image. The formula for obtaining the modified image is the same as above.

  In order to accurately map lens non-uniformities, the calibration standard 601 must completely block the lens aperture. However, standard 601 can be much smaller because standard 601 is much closer to lens 107 than the standard placed on the sample surface in the previous system. This relationship is shown in FIG. The focusing aperture is indicated by the dotted line 701. As shown, the standard 601 is approximately the same size as the lens 107 because it is close to the lens. However, the standard 703 placed on the sample surface must be much larger to plug the lens aperture, increasing manufacturing costs and difficulty.

  While the present invention can be used to overcome non-uniformities associated with lenses and detectors, the illumination source must be uniform to quantify the sample image. If the source is not uniform, the inaccuracy associated with the sample image, I1 (x, y), is not completely eliminated by the technique of the present invention. Illumination uniformity can be achieved using multiple light sources, reflectors, masks or scanning light systems.

  However, light uniformity is not required during the calibration step of embodiments of the present invention. Although increasing light uniformity increases the accuracy of the described calibration technique, considering the distance 605 from standard 601 to light source 103, illumination uniformity is not a requirement of this technique.

  FIG. 8 shows the relationship between light uniformity and calibration standard placement. The light source 801 illuminates a calibration standard 803 that is placed in close proximity to the light source 801. As defined by Lambert's law, the intensity of an incremental region with a small source J θ is the intensity of the incremental region in the normal direction, Jo, multiplied by the cosine of the angle θ measured from the surface normal. equal. Therefore, the intensity measured at a point 805 on the standard 803 directly above the area 807 of the source 801 is equal to the intensity of the area 807. In contrast, the intensity measured at point 809 at the opposite end of standard 803 is greatly reduced. For example, considering an angle θ of 80 degrees, the intensity at point 809 is only 17 percent of the intensity measured at point 805. Thus, if the source 801 is non-uniform, the calibration standard 803 is illuminated non-uniformly and the calibration technique is not only a function of lens and detector non-uniformity, but also a function of illumination non-uniformity.

  In contrast, using a calibration standard some distance from the source 801, the illumination non-uniformity has little impact on the calibration technique. For example, assuming that a 2 centimeter diameter measurement calibration standard 811 is from source 801 to 65 centimeters, the difference in intensity measured at points 813 and 815 is less than 1 percent. Therefore, placing the calibration standard close to the lens and away from the source can greatly reduce the effects of illumination non-uniformity.

  FIG. 9 is a diagram of another embodiment of the present invention. In this embodiment, a diffuser 901 is added to the same elements as shown in FIG. The diffuser 901 is attached between the calibration standard 601 and the lens 107. The diffuser 901 also smoothes out small non-uniformities within the calibration standard so that the system only monitors lens and detector non-uniformities.

  FIG. 10 is a detailed diagram of an embodiment of the present invention. In this example, the sample 1001 is illuminated with a light source 1003. The source 1003 is composed of three light bulbs 1005 arranged in the tray 1007. The tray 1007 rides on a pulley and belt system 1009 controlled by the motor 1011. During sampling, the light tray 1007 scans the sample 1001 starting at point 1013 and ending at point 1015. The scan speed, source intensity, and intervening filter 1017 will determine the fluorescence of an appropriately marked region of the sample 1001 to determine the intensity of the radiation that illuminates the sample 1001. The illumination is made uniform by the scanning source 1003.

  The fluorescence emitted from the sample 1001 passes through the aperture 1019 or the filter 1019 of the filter wheel 1021 before being imaged by the lens assembly 1023 to the detector 1025. In this embodiment, detector 1025 is a CCD detector, preferably a two-dimensional array. If a calibration image is required, the filter wheel 1021 rotates to place the calibration standard 1027 in front of the lens assembly.

  There are several ways to illuminate the calibration standard 1027. First, radiation passing from the source 1003 through the sample 1001 can be used. Due to the pattern on the gel sample 1001, the illumination intensity is very non-uniform. However, as indicated above, this standard is relatively insensitive to illumination non-uniformity. The source 1003 can be used in a normal scanning mode or held in a stationary position in the center of the device. Second, the sample 1001 can be removed and the source 1003 can be used in scanning or stationary mode. Third, another source 1029 can be used to illuminate the calibration standard 1027. It is desirable to position the source 1029 on the sample surface as shown so that the sample 1001 does not need to be removed prior to obtaining a calibration image. In an embodiment, a set of sources 1029 is used to illuminate standard 1027, improving the uniformity of light reaching standard 1027.

  FIG. 11 is a close-up view of the detector assembly 1025, the lens assembly 1023, and the filter wheel 1021. As shown, the calibration standard 1027 is rotated to a plane where calibration measurements can be made.

  FIG. 12 is a block diagram illustrating the process steps of the present invention. Initially, the sample is placed in the test apparatus (step 1201). Although the present invention is not limited to gel electrophoresis applications, it is one of the most commonly used applications. In this example, the sample is a gel, and certain regions, structures or molecules are labeled with a fluorescent marker that fluoresces at a specific wavelength when emitted at the excitation wavelength of the marker. Since the emission wavelength is different from the excitation wavelength, the emission can be distinguished from the source due to the use of a filter. Radiation from the selected area is imaged on a detector to form an image of the sample (step 1203). Once the sample image is acquired, the calibration standard is moved (step 1205). In order to correctly measure the non-uniformity of the lens and detector assembly, the aperture and magnification settings of the system have not been changed from those used during the sample imaging step. A calibration image is acquired using a sample illumination source or the alternative sources described above (step 1207). Then, the camera shutter is closed (step 1209), and a dark field image is acquired (step 1211). Then, the sample image is corrected using a well-known mathematical relationship such as the above correction algorithm (1213).

  FIG. 13 is a diagram of the main components of a system according to an embodiment of the present invention. In this embodiment, the central system component is connected to processor 1301. The processor 1301 can automate the entire process, making the correction process linear and minimizing the risk of user error. To use this system, the sample 1001 is first placed in the electrophoresis apparatus 1303. The user then manually sets the aperture and magnification of the system unless the system is designed to automatically select these parameters. In the embodiment, the user inputs a marker (eg, fluorescence) to be used for the sample 1001 to the processor 1301 via the user interface 1305. Based on this information, the processor 1301 uses an integrated lookup table to determine appropriate excitation and emission settings. Then, the processor 1301 moves an appropriate filter by operating the filter wheel 1021.

  Once the system operating parameters are set manually or automatically, the user starts execution by sending a corresponding signal to the processor 1301 via the interface 1305. Then, the processor 1301 turns on the source 1003 and acquires an image of the sample 1001. When the source 1003 is scanning the source as shown in FIG. 10, the processor 1301 also controls the scanning operation by controlling the scanning motor 1011. The image acquired by the detector 1025 is stored in a memory resident in the processor 1301 and displayed on the monitor 1307. When the detector 1025 is a CCD camera, data is easily stored in a digital format, so that subsequent data manipulation is facilitated. Depending on the configuration of the processor 1301, several actions may occur next. If the system is in fully automatic mode, the system will automatically begin the sample image correction process. In this mode, the initial uncorrected image may or may not be displayed depending on the system setup. An advantage of displaying the initial image is that the user can determine that the cycle is complete before applying the correction technique because the image contains no useful data.

  In automatic mode, after obtaining a sample image, processor 1301 turns off source 1003 and moves the calibration standard. Since the calibration standard is in the filter wheel 1021, the processor 1301 simply rotates the wheel until the appropriate filter recess is in place. The processor 1301 then activates an appropriate illumination source of the source 1003 or the alternative source 1029 and acquires a flat field image. Data from this image is stored in resident processor memory. When the calibration standard image is complete, the processor 1301 closes the shutter of the camera assembly 1025 and acquires a dark field image. Using these two additional images, the processor 1301 corrects the sample image and displays the corrected image on the monitor. Both uncorrected and corrected images and flat and dark fields can be retrieved and stored for later use.

  FIG. 14 is an illustration of another method of determining lens assembly non-uniformity. The sample surface 1401 is illuminated with a light source 1403. In this embodiment, the light source 1403 is comprised of a series of light bulbs 1405, but this technique applies equally to other light sources. In normal use, a sample such as an electrophoresis gel is in the sample surface 1401. Fluorescence from the excited region of such sample is imaged to detector 1409 by lens assembly 1407.

  A secondary source 1411 is used to characterize the lens assembly 1407. Source 1411 is a line source, such as a series of light emitting diodes (LEDs) 1413. The source 1411 can also consist of a series of other types of point sources or a line source such as a long light bulb. Radiation from source 1411 is magnified by lens 1415 before being reflected by mirror 1417. Since mirror 1417 is a partial reflector, it can remain in place during normal operation of the system. The mirror 1417 can also be dedicated to the measurement work of lens non-uniformity. If mirror 1417 is a dedicated mirror, it is placed on the stage so that it can be moved out of the sample imaging path during normal system operation.

  Mirror 1417 reflects radiation from source 1411 along path 1419. This radiation then passes from the lens assembly 1407 to the detector 1409. Given the uniform detector response (such as sensitivity) that can be obtained with a CCD array, and also the uniformity of mirror 1417, lens 1415, and source 1411, if there is any non-uniformity measured by detector 1409, lens assembly 1407 Is due to.

  The apparatus shown in FIG. 14 assumes that the lens assembly 1407 exhibits spherical symmetry, so that lens non-uniformity can only be determined along one axis 1421. If the lens assembly 1407 does not exhibit such symmetry, further non-uniformity measurements must be made along the other axis.

  Another way to characterize the non-uniformity of the lens assembly is to remove the lens assembly and characterize it separately from the electrophoresis apparatus. For example, in the system shown in FIG. 15, the lens assembly 1501 is removed from the electrophoresis apparatus (not shown) and placed between the source 1503 and the detector 1505 of the calibration bench 1507. In this environment, a very uniform source 1503 and detector 1505 can be used, thus ensuring high characterization accuracy. Once characterized, the lens assembly 1501 is reattached to the electrophoresis apparatus.

  FIG. 16 is a diagram of the main components of the system according to the invention. In this embodiment, the central system component is coupled to a processor 1601 that is connected to a user interface 1603. The processor 1601 is necessary to perform the interpolation and normalization process in a timely manner. This embodiment can be used regardless of the method used to characterize the non-uniformity of the lens assembly 1605.

  To obtain a sample image, the source 1607 is turned on to fluoresce the appropriately marked region of the sample 1609. The fluorescence is imaged on the detector 1611 by the lens assembly 1605. The sample image is immediately displayed on the display monitor 1613 or stored as a sample file in a memory associated with the processor 1601.

  To correct the sample image for lens non-uniformity, the lens aperture and magnification settings used during sample image acquisition must be input to the processor 1601. These settings are necessary to allow the processor 1601 to determine the appropriate modification file to use during the normalization process. After normalizing the image file with an appropriate modified file, the modified image file can be displayed on the monitor 1613. The modified image file can also be stored in memory for later retrieval and use.

  There are many ways to obtain lens settings by the processor 1601. The easiest way for the user is to manually enter the configuration information using the interface 1603. The second method is to automatically determine the lens settings by the processor 1601. In this embodiment, the lens 1605 includes one or more setting sensors 1615. Sensor 1615 is coupled to processor 1601 by line 1617. The sensor 1615 is mechanical or electro-optical and employs a well-known technique.

  FIG. 17 is an illustration of another technique that utilizes one calibrated light source to determine the aperture and magnification settings of the lens 107. The light source may be a light emitting diode (LED) or other source type that can be easily calibrated. The source can be placed at location 1701 or location 1703 outside a stationary field. If the source is within the field of view of the imager, such as location 1701, it must be removable. For example, the source can be moved using stage 1705.

  To determine the lens settings using this method, place a calibrated source and turn it on. Acquire the source image using the same settings used to acquire the image of the sample in question. The magnification setting is determined by looking at the image size of the calibrated source, while the aperture setting is determined by the intensity of the source.

  FIG. 18 is a diagram of another embodiment of the present invention. The sample surface 1801 is illuminated with a light source 1803. In this embodiment, the light source 1803 is comprised of a series of light bulbs 1805, but the invention is equally applicable to other light sources. In normal use, a sample such as an electrophoresis gel is within the sample surface 1801. Fluorescence from the excited region of such sample is imaged to detector 1809 by lens assembly 1807. The detector 1809 is preferably a two-dimensional CCD array.

  To remove non-uniformities arising from the illumination source 1803, a mirror 1811 is used to sample a portion of the source. In one embodiment of the invention, mirror 1811 is coated with a partial reflector so that some of the light from source 1803 reaches lens 1807 and detector 1809. In this example, a second portion of light from source 1803 is reflected along path 1813. Assuming that only a small portion of light is reflected by the mirror 1811 along the path 1813, one of the main advantages of this embodiment is that the mirror 1811 need not be removed during normal operation of the system. However, in this configuration, the mirror 1811 introduces non-uniformity, which must also be taken into account in the correction process. In an alternative embodiment, mirror 1811 is coupled to a stage (not shown). During normal operation, the stage removes the mirror 1811 from the light path so that it does not interfere with the light path from the sample on the plane 1801 to the imaging lens assembly 1807. In order to determine the non-uniformity of the light source 1803, the stage moves the mirror 1811 to a sampling position as shown in FIG.

  To determine the non-uniformity of the illumination source 1803, the mirror 1811 reflects a portion of the source 1803 along the path 1813 through the lens 1815. The lens 1815 focuses the light on the linear detector array 1817. Detector 1817 is preferably a linear CCD array. However, other types of in-line detector arrays can be used. Further, the array 1817 need not be continuous. Thus, the array 1817 can be composed of a series of point detectors and a uniformity profile between the point detectors interpolated from the measured points.

  The dimensions and positions of mirror 1811, lens 1815, and detector 1817 are determined by the symmetry of source 1803. For example, a source such as that shown in FIG. 18 can be expected to be relatively uniform along axis 1819 due to the illumination uniformity of bulb 1805 extending along each major axis. In contrast, source 1803 can be expected to have an illumination profile along axis 1821 similar to that shown in FIG. Therefore, by measuring the profile of the source 1803 along the axis 1821 as shown, the non-uniformity of the entire source can be determined.

  Once the source non-uniformity is determined, the sample image can be corrected. FIG. 19 is a block diagram outlining the main steps of correcting an image using the present invention. First, the source profile must be measured using the techniques described above (step 1901). Once the profile is determined, the modified file is stored in memory associated with the system image processor (step 1903).

The modified file must contain as many modified data points as there are points from the sample imaging process. Since the number of modified data points is generally much less than the number of sample points, the modified data points must be filled by the processor. The correction data points entered take into account both the symmetry of the source and the number of pixels of the detector 1817. FIG. 20 illustrates the interpolation process. Matrix 2001 is represented by an 11 × 11 matrix of modified data points, thereby reproducing a sample image of 121 sample data points. Assuming a source as shown in FIG. 18 that is uniform along axis 1819 and non-uniform along axis 1821, most of the modified data points can be entered by the processor. In the illustrated example, the modified data point 2003 in column 2005 has been measured using the present invention. The modified data point 2007 in column 2005 is filled in by interpolation. Given the uniformity of the source along axis 1819, the remaining column 2009 is simply a reproduction of column 2005.

  When the source profile is measured, a sample image is acquired (step 1905) and stored (step 1907). Prior to normalizing the image, in an embodiment, the modified file is first smoothed using a smoothing function as is well known in the art (step 1909). By this smoothing function, if there is a noise source of the corrected file that makes the corrected sample image defective, it is not amplified by the normalization process. Finally, in order to realize the corrected sample image file, normalization is performed by dividing the sample file by a correction file (or a smoothed correction file if step 1909 is applied) (step 1911). The modified sample image file can be displayed (step 1913) or stored (step 1915) for later use.

  FIG. 21 is a diagram of the main components of a system according to another embodiment of the present invention. In this embodiment, the central system component is connected to a processor 2101 that is connected to a user interface 2102. A processor 2101 is required to perform the interpolation / normalization process in a timely manner, but this can also automate much of the process. In use, sample images can be acquired and stored before or after profiling the illumination source. Furthermore, since the source is relatively constant for a short period of time, it is not necessary to profile the source with each sample run. However, correction accuracy is improved by profiling the source at each sample run. In an embodiment, the source is profiled only periodically and the new profile is compared to the last profile to determine whether there has been a change, such as a change over time of the bulb.

  Assuming that the sample image is acquired before the source profile, the sample 2103 is first placed in the electrophoresis apparatus 2105. In this embodiment, the source profile mirror 2107 is attached to the retractable stage 2109. During the sample run, the stage 2109 is pulled and the mirror 2107 is at position 2111.

  To obtain a sample image, the source 2113 is turned on to fluoresce an appropriately marked area of the sample 2103. The fluorescence is imaged on the detector 2117 by the lens assembly 2115. The sample image is stored as a sample file in a memory associated with the processor 2101. If desired, the sample image can be displayed on the monitor 2119 before modification.

  To obtain the source profile, the sample 2103 is removed from the apparatus 2105 and the mirror 2107 is moved on the stage 2109. The source 2113 is turned on, and the source profile is imaged on the detector array 2121 by the lens 2123. If necessary, an additional source file can be acquired based on the symmetry of the source 2103. For example, by replicating the mirror 2107, the lens 2123, and the detector 2121 along the second axis, the second source profile can be obtained along the first perpendicular axis. Once the source profile or profiles are obtained, the processor 2101 can create a modified file and, if desired, a smoothed modified file. This data is then used to normalize the image as described above.

  In another embodiment of the invention, the lens assembly can be profiled using a source profiling mirror. This embodiment is shown in FIG. This embodiment can be used with the previously described embodiment to obtain a sample image modified for source and lens non-uniformities. If the source is a uniform source, this embodiment can be made separate to correct non-uniformities in the lens assembly only. The source can be made uniform using a wide range of techniques such as scanning sources, reflectors and / or masks.

  To remove non-uniformity due to the lens assembly 1807, the secondary source 2201 is used. Source 2201 is a line source, such as a series of light emitting diodes (LEDs) 2203. Source 2201 can also consist of a series of other types of point sources or a single line source such as a long bulb. Radiation from source 2201 is magnified by lens 2205 before being reflected by mirror 1811. As in the previous embodiment, mirror 1811 is a partial reflector so that it can remain in place during normal operation of the system. The mirror 1811 can also be dedicated to measuring the non-uniformity of the lens and / or illumination source. If mirror 1817 is a dedicated mirror, it is placed on the stage so that it can be moved out of the sample imaging path during normal system operation.

  Mirror 1811 reflects radiation from source 2201 along path 2207. The radiation then passes through the lens assembly 1807 to the detector 1809. Assuming a uniform detector response (such as sensitivity) that can be obtained with a CCD array, and assuming uniformity of mirror 1811, lens 2205, and source 2201, the non-uniformity measured by detector 1809 is due to lens assembly 1807. is there.

  Since the apparatus shown in FIG. 22 assumes that the lens assembly 1807 exhibits spherical symmetry, lens non-uniformity can only be determined along one axis 2209. If the lens assembly 1807 does not exhibit such symmetry, further non-uniformity measurements must be made along the other axis.

  Once the lens non-uniformity is determined, the sample image can be corrected according to the same steps outlined in FIG. The only difference is that instead of measuring the source profile, step 1901 measures the profile of the lens assembly 1807. The rest of the steps, such as creating a modified file and then using the modified file to normalize the sample file are the same as described above.

  FIG. 23 is a diagram of a system according to the present invention, including the components necessary to perform a lens non-uniformity measurement. This figure is the same as that shown in FIG. 21 except that a source 2201 and a lens 2205 are added. In this embodiment, the source 2201 is connected to the processor 2101 so that lens non-uniformity measurements can be automated.

  FIG. 24 is a block diagram outlining the major steps used in accordance with another embodiment of the present invention. In this embodiment, the source profile is first determined using the system described above (step 2401). Once the source profile is determined, the secondary source profile can be changed to mimic the measured profile of the actual source (step 2403). This step requires that the source 2201 be composed of a series of point sources 2203 that can be individually controlled. The point source 2203 is preferably calibrated in advance and controlled by the processor 2101. Since the source 2201 is tuned to provide the same profile as the source 2403, the stored correction file takes into account both the non-uniformity of the illumination source 2403 and the non-uniformity of the lens assembly 2407. As in the previous embodiment, the modified file must contain the same number of modified data points as the number of data points from the sample imaging process. As such, the modified file contains a number of modified data points that have not been measured, but have been calculated by the processor 2101 using standard interpolation functions.

  After measuring the modified processor file and storing the appropriate modified data file, a sample image is acquired (step 2409), stored (step 2411), and normalized (step 2413). Similar to the previous embodiment, if necessary, a smoothing function can be applied to the modified data file before it is used to normalize the sample image. Finally, the modified sample file is stored for later use (step 2415) or immediately displayed (step 2417).

  As will be appreciated by those skilled in the art, the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. For example, the present invention is not limited to specific excitation and emission wavelengths. Therefore, a UV excitation marker that emits fluorescence at a visible wavelength can be used. Other techniques for lens non-uniformity characterization can also be used. Accordingly, the disclosure and description herein are for purposes of illustration and do not limit the scope of the invention described in the following claims.

It is sectional drawing of the gel electrophoresis apparatus by a prior art. It is the intensity profile of one bulb measured perpendicular to the bulb axis. FIG. 6 is an intensity profile of a source with multiple valves. It is a figure which shows the method of improving the uniformity of an illumination source. FIG. 4 is a block diagram outlining the basic steps of correcting an image for lens non-uniformity according to the present invention. It is a figure which shows the Example of this invention. It is a figure which shows the relationship between a lens aperture and the size of a calibration standard. It is a figure which shows the relationship between the uniformity of light, and a calibration standard. FIG. 6 illustrates another embodiment of the present invention utilizing a diffuser with a calibration standard. It is detail drawing of the Example of this invention. FIG. 11 is a close-up view of the detector assembly, lens assembly, and filter wheel of the embodiment shown in FIG. 10. FIG. 3 is a block diagram illustrating process steps of the present invention. FIG. 2 shows the main components of a system according to a preferred embodiment of the present invention. FIG. 6 illustrates another method of determining lens assembly non-uniformity. FIG. 6 illustrates another method of determining lens assembly non-uniformity. FIG. 2 shows the main components of the system according to the invention. FIG. 5 shows a method used to determine the aperture and magnification settings of a lens assembly. It is a figure which shows the Example of this invention. FIG. 6 is a block diagram outlining the main steps used when modifying an image for illumination non-uniformity according to the present invention. It is a figure which shows the interpolation process used for a correction file preparation. FIG. 2 shows the main components of a system according to a preferred embodiment of the present invention. FIG. 5 shows another embodiment of the present invention that can be used to measure non-uniformity of both the illumination source and the lens assembly. FIG. 2 shows a system according to the invention including the components necessary to perform lens non-uniformity measurements. FIG. 6 is a block diagram outlining the main steps used by another embodiment of the present invention.

Claims (9)

A platform for holding the electrophoresis gel;
A first light source for illuminating the electrophoresis gel;
A first lens assembly for imaging radiation from a labeled region of the electrophoresis gel onto a first detector;
An intensity pattern of a second light source passing through the first lens assembly is measured by the first detector to determine a first lens assembly profile. The first detector is used to determine the first lens assembly profile. A mirror for reflecting illumination from two light sources;
A processor coupled to the first detector for normalizing an image of the electrophoresis gel using the first lens assembly profile;
An electrophoretic system comprising:   The electrophoresis system according to claim 1, wherein the second light source is a line source.   The electrophoresis system according to claim 2, wherein the line source comprises a plurality of individual light sources.   The electrophoresis system according to claim 1, further comprising a second lens assembly interposed between the second light source and the mirror.   The electrophoresis system according to claim 4, wherein the second lens assembly is an image magnifier. A portion of the light from the first light source array is reflected to the detector array via the first lens assembly;
Measuring an intensity pattern with the detector array of the first light source array;
Determining a first lens assembly correction file from the measured intensity pattern;
Storing the first lens assembly correction file;
Illuminating the electrophoretic gel with an electrophoretic gel illumination source, at least one labeled region of the illuminated gel fluoresces,
Imaging the illuminated electrophoresis gel on a second detector array;
Store electrophoresis gel image file,
Normalizing the electrophoresis gel image file with the first lens assembly modification file;
Storing the normalized electrophoresis gel image file;
A method for correcting an electrophoretic gel image, comprising each step.   The method of claim 6, comprising displaying the normalized electrophoretic gel image file.   The method of claim 6, comprising enlarging the portion of light from the first light source array with a second lens assembly prior to the reflecting step. By moving a mirror to a first position, the mirror in the first position performs the reflection step;
The method of claim 6, comprising moving the mirror to a second position so that the mirror in the second position illuminates the electrophoretic gel with the electrophoretic gel illumination source without interference.

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