JP2007024604A - Fluorescence measuring instrument and fluorescence measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence measuring instrument capable of suppressing the occurrence of a cross talk when the fluorescences generated from a plurality of samples are measured at the same time. <P>SOLUTION: An exciting light feed part 10 is constituted so as to feed exciting light to a plurality of samples and a discrimination part 40 judges whether the quantity of fluorescence detected by a light detecting part 20 exceeds a fluorescent threshold value TL2. An operation part 50 calculates the quantity of fluorescence from the quantity of exciting light with respect to fluorescence of which the quantity exceeds the fluorescent threshold value TL2. In the exciting light feed part 10, the feed of exciting light to the sample discharging fluorescence of which the quantity exceeds the fluorescence threshold value TL2 is stopped by the indication from a timing controller 32. Further, the quantity of the exciting light fed to the sample discharging fluorescence of which the quantity does not exceed the fluorescence threshold value TL2 is increased to continuously perform measurement. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fluorescence measuring apparatus and a fluorescence measuring method.

  As a fluorescence measuring device that measures fluorescence generated from a plurality of samples, such as a DNA chip, there is an optical reading device described in Patent Document 1, for example, or a light measuring device described in Patent Document 2. In the optical reading device described in Patent Literature 1, excitation light is supplied to each of a plurality of samples at different times to generate fluorescence, and fluorescence generated by one light detection device is detected. Yes.

In the light measurement device described in Patent Document 2, excitation light is supplied to the entire plurality of samples by moving the sample to be measured and changing the supply region of the excitation light, and fluorescence is measured.
JP-A-11-313698 JP 2004-279258 A

  However, in the optical reading device described in Patent Document 1, since a plurality of samples are measured by supplying excitation light in a plurality of times for each sample, it is proportional to an increase in the number of samples. Therefore, it takes a long time to measure.

  On the other hand, Patent Document 2 describes a method of simultaneously measuring fluorescence generated from a plurality of samples using a CCD camera. However, when simultaneously measuring fluorescence generated from a plurality of samples using a CCD, when measuring the fluorescence by associating a CCD pixel group (or pixel) with each sample, it should correspond to different neighboring samples. The pixel group (or pixel) is also exposed to light, and the problem of crosstalk that affects the measurement of different neighboring samples occurs. The problem of crosstalk occurs not only when a CCD camera is used, but also when other photodetectors such as photomultiplier tubes are used. Patent Document 2 does not discuss the suppression of crosstalk.

  The present invention has been made to solve the above problems, and provides a fluorescence measuring apparatus and a fluorescence measuring method capable of suppressing the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples. The purpose is to do.

  In order to achieve such an object, a fluorescence measuring apparatus according to the present invention includes an excitation light source, excitation light supply means for supplying excitation light output from the excitation light source to each of a plurality of samples, and a plurality of A light detection means for detecting fluorescence emitted from the sample, and a discrimination means for discriminating whether or not the amount of fluorescence emitted from each of the plurality of samples and detected by the light detection means exceeds a predetermined fluorescence threshold value; In the discriminating means, the calculating means for performing processing for calculating the light quantity of the fluorescence discriminated as having exceeded the fluorescence threshold by the discrimination means based on the light quantity of the excitation light supplied to the sample that has emitted the fluorescence Discontinue the supply of excitation light to the sample that has emitted the fluorescence that has been discriminated if the light quantity exceeds the fluorescence threshold, and the sample that has emitted the fluorescence that has been discriminated if the light quantity does not exceed the fluorescence threshold by the discrimination means It characterized in that it and a pumping light controlling means for increasing the amount of the excitation light to be fed.

  In this fluorescence measurement apparatus, excitation light is supplied to each of a plurality of samples, and the amount of fluorescence emitted from each sample (hereinafter simply referred to as fluorescence amount) is changed to the amount of excitation light supplied to each of the plurality of samples ( Hereinafter, the calculation is simply based on the excitation light amount. Therefore, fluorescence emitted from a plurality of samples can be measured at the same time, and the time required for measurement can be shortened. In addition, the light quantity here means the amount of energy of light. In addition, this fluorescence measuring device discriminates whether or not the fluorescence amount exceeds a predetermined fluorescence threshold value in the discrimination means, and calculates the fluorescence amount based on the excitation light amount only for those exceeding the fluorescence threshold value by the calculation means. is doing. In addition, the supply of excitation light is stopped for samples whose fluorescence amount exceeds the fluorescence threshold, and the fluorescence amount is continuously measured only for samples whose fluorescence amount does not exceed the fluorescence threshold. Become. Therefore, even when there is a large difference in the amount of fluorescent light generated from adjacent samples, it is possible to suppress the occurrence of crosstalk. Thus, this fluorescence measuring apparatus can suppress the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples. For the sample whose fluorescence amount does not exceed the fluorescence threshold, the amount of excitation light supplied is increased. Therefore, it is possible to measure a weak fluorescence amount while suppressing the influence of crosstalk. In addition, since it is possible to measure even weak fluorescence while suppressing the influence of crosstalk, the sensitivity range (dynamic range) that can be measured by the apparatus can be increased.

  The excitation light control means may increase the amount of excitation light, for example, by changing the power supplied to the excitation light source. In addition, for example, there are a plurality of excitation light sources, which correspond to at least a plurality of samples on a one-to-one basis, and the excitation light control means changes the power supplied to the excitation light source and outputs the excitation light from the excitation light source. The supply of excitation light may be stopped by canceling.

  Alternatively, for example, a light modulation unit that modulates the excitation light output from the excitation light source may further be provided, and the light intensity of the excitation light may be increased by modulating the intensity of the excitation light in the light modulation unit. . In addition, for example, it further includes a light modulation unit that modulates the excitation light output from the excitation light source, and the excitation light is supplied to a plurality of samples by modulating the intensity of the excitation light in the light modulation unit. You may cancel.

  For example, the plurality of samples may be arranged two-dimensionally. Even in this case, fluorescence emitted from a plurality of samples can be measured simultaneously while suppressing the occurrence of crosstalk.

  The excitation light source is preferably a semiconductor light emitting element. By applying the semiconductor light emitting element, the amount of excitation light can be easily controlled, and in addition, the apparatus can be downsized and the power consumption can be reduced.

  The excitation light supply means preferably further includes a light guide that guides the excitation light from the excitation light source to each of the plurality of samples. In this case, excitation light can be easily supplied to each of the plurality of samples.

  The light detection means is preferably a solid-state image sensor. In this case, it is possible to simultaneously measure fluorescence generated from a large number of samples.

  The calculation means calculates the amount of fluorescence by dividing the amount of fluorescence discriminated as having exceeded the fluorescence threshold by the discrimination means by the amount of excitation light supplied to the sample that has emitted the fluorescence. Also good. Even if the supply amount of excitation light is changed, it is possible to easily measure the fluorescence generated from a plurality of samples.

  The fluorescence measurement method according to the present invention includes an excitation light supply step for supplying excitation light output from an excitation light source to each of a plurality of samples, a light detection step for detecting fluorescence emitted from the plurality of samples, A discrimination step for discriminating whether or not the amount of fluorescence emitted from each of the plurality of samples and detected in the light detection step exceeds a predetermined fluorescence threshold, and discriminating if the amount of light exceeds the fluorescence threshold by the discrimination means And calculating the amount of fluorescent light based on the amount of excitation light supplied to the sample that has emitted the fluorescent light, and the fluorescence discriminated as having exceeded the fluorescence threshold in the discrimination step. Supply to the sample that emitted the fluorescence that was discriminated if the light intensity did not exceed the fluorescence threshold in the supply stop step and the discrimination step to stop supplying the excitation light to the released sample An excitation light amount increasing step of increasing the amount of excitation light that, characterized in that it comprises a.

  In this fluorescence measurement method, excitation light is supplied to each of a plurality of samples, and the amount of fluorescence emitted from each sample is calculated based on the amount of excitation light supplied to each of the plurality of samples. Therefore, fluorescence emitted from a plurality of samples can be measured at the same time, and the time required for measurement can be shortened. Further, in this fluorescence measurement method, it is discriminated whether or not the fluorescence amount exceeds the fluorescence threshold value in the discrimination step, and only the fluorescence amount exceeding the fluorescence threshold value is calculated in the calculation step. In addition, the supply of excitation light is stopped for samples whose fluorescence amount exceeds the fluorescence threshold, and the fluorescence amount is continuously measured only for samples whose fluorescence amount does not exceed the fluorescence threshold. Become. Therefore, even when there is a large difference in the amount of fluorescent light generated from adjacent samples, it is possible to suppress the occurrence of crosstalk. Thus, this fluorescence measurement method can suppress the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples. In addition, the amount of excitation light to be supplied is increased for samples whose fluorescence amount does not exceed the fluorescence threshold. Therefore, it is possible to measure a weak fluorescence amount while suppressing the influence of crosstalk. In addition, since it is possible to perform measurement while suppressing the influence of crosstalk up to weak fluorescence, the sensitivity range (dynamic range) that can be measured by this measurement method can be increased. Note that the order of each step may be changed as appropriate.

  According to the present invention, it is possible to provide a fluorescence measuring apparatus and a fluorescence measuring method capable of suppressing the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples.

  Hereinafter, preferred embodiments of a fluorescence measuring apparatus and a fluorescence measuring method according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. The dimensional ratios in the drawings do not necessarily match those described.

(First embodiment)
FIG. 1 is a configuration diagram schematically showing a fluorescence measuring apparatus according to the first embodiment. As shown in FIG. 1, the fluorescence measuring apparatus 1 according to the present embodiment includes an excitation light supply unit 10 that supplies excitation light to each of a plurality of samples, and a light detection unit that detects fluorescence emitted from the plurality of samples. 20, a timing controller 32 that controls excitation light supplied to a plurality of samples, a discrimination unit 40 that discriminates whether or not the amount of fluorescence exceeds a fluorescence threshold, and an operation that performs processing for calculating the amount of fluorescence Part 50. Here, the amount of light refers to the amount of light energy.

  The excitation light supply unit 10 in this embodiment includes a plurality of semiconductor light emitting elements (LEDs) 11, an LED power controller 12, and an optical fiber 13. FIG. 2 shows the relationship among a plurality of samples S 1 whose fluorescence is measured by the fluorescence measuring apparatus 1, the semiconductor light emitting element 11, and the optical fiber 13. The plurality of samples S1 are arranged on the plate P1 for measurement. The plate P1 is a microtiter plate, and a plurality of wells are formed in a two-dimensional array on one main surface of the plate P1. Each of the plurality of samples S1 is stored in each well on the plate P1. The number of wells may be the same as or more than that of the sample S1, but in this embodiment, the number of wells is the same as the number of the sample S1.

  The plurality of semiconductor light emitting elements 11 function as an excitation light source that supplies excitation light to each of the plurality of samples S1. Further, the plurality of semiconductor light emitting elements 11 are arranged in a two-dimensional array like the plurality of wells formed on the plate P1 so that each semiconductor light emitting element 11 corresponds to each of the plurality of samples S1 one to one. Yes. Note that the number of the semiconductor light emitting elements 11 may be at least one-to-one with the sample S1, and may be the same as or more than the number of the samples S1.

  The LED power controller 12 can independently control the power supplied to each of the plurality of semiconductor light emitting elements 11 based on an instruction from a timing controller 32 described later. When the power supplied to the semiconductor light emitting element 11 is changed in the LED power controller 12, the amount of excitation light is changed.

  There are a plurality of optical fibers 13, and the same number as the plurality of samples S <b> 1 (wells). As shown in FIG. 2, each optical fiber 13 functions as a light guide that guides excitation light from each semiconductor light emitting element 11 to each sample S1. By supplying excitation light through the optical fiber 13, each sample S1 emits fluorescence.

  Returning to FIG. 1 again, the description of the fluorescence measuring apparatus 1 will be continued. The light detection unit 20 detects fluorescence emitted from the plurality of samples S1. The light detection unit 20 in the present embodiment includes a CCD camera 21, a camera controller 22, and an A / D converter 23. On the light receiving surface of the CCD camera 21, fluorescent images are formed by fluorescence emitted from the plurality of samples S1. The fluorescence image is converted into an electrical signal by the CCD camera 21 and fluorescence is detected. Here, since the plurality of samples S1 are arranged in a two-dimensional array, the fluorescence image is detected as a two-dimensional image.

  The CCD camera 21 and the camera controller 22 are connected by a signal line. The camera controller 22 controls the operation of the CCD camera 21.

  The electrical signal obtained by the CCD camera 21 is sent to the A / D converter 23. The A / D converter 23 performs A / D conversion on an electrical signal that is an analog signal, and generates a video signal that is a digital signal. The A / D converter 23 outputs a video signal indicating the amount of fluorescence detected by the light detection unit 20 to the video signal level detector 31.

  In the video signal level detector 31, a video signal threshold TL1 is set. The video signal level detector 31 sends a signal to the timing controller 32 when the level of the video signal exceeds the video signal threshold value TL1.

  The timing controller 32 receives a signal from the video signal level detector 31, and instructs the LED power controller 12 about power supplied to the semiconductor light emitting element 11 (for example, an instruction signal for making the supplied power constant). Send. The timing controller 32 functions as excitation light control means as will be described later, and in the present embodiment, outputs from the respective parts constituting the fluorescence measuring apparatus 1 are input and instructions are given to other parts according to the input values. It also functions as a control means for outputting a signal.

  The discriminating unit 40 in the present embodiment includes an image processor 41, a dot area adder 42, and a fluorescence amount discriminator 43. The image processor 41 includes a frame memory that takes in and stores the video signal output from the video signal level detector 31 as an input. In the frame memory of the image processor 41, the accumulated video signal is stored as image data. The image processor 41 outputs the image data stored in the frame memory to the dot area adder 42. Further, the image processor 41 sends a capture end signal to the timing controller 32 when the capture of the video signal from the video signal level detector 31 is completed.

  The dot area adder 42 receives image data stored in the frame memory of the image processor 41 as input. The dot area adder 42 receives the instruction signal from the timing controller 32 that has received the capture end signal, and obtains a dot area addition value obtained by adding the pixel data in each dot area from the image data. Here, the dot area refers to an area on the image data corresponding to the well portion formed on the plate P1 including each sample S1. The dot area addition value is a value obtained by adding all the pixel data in each dot area. Therefore, the dot area addition value corresponds to the amount of fluorescent light emitted from the sample S1 stored in the well portion of the plate P1 corresponding to the dot area for which the dot area addition value was obtained.

  The dot area adder 42 outputs the dot area addition values thus obtained to the fluorescence amount discriminator 43 in association with the addresses of the semiconductor light emitting elements 11 arranged in a two-dimensional array. Here, the address of the semiconductor light emitting element 11 is a pointer that specifies the position of the semiconductor light emitting element 11 arranged in a two-dimensional array, for example, by indicating the number of columns and the number of rows. Further, the address of the semiconductor light emitting element 11 corresponding to the dot area addition value refers to the address of the semiconductor light emitting element 11 that supplies excitation light to the sample S1 corresponding to the dot area for which the dot area addition value has been obtained. .

  The fluorescence amount discriminator 43 includes a dot memory. The fluorescence amount discriminator 43 stores the dot area addition value output from the dot area adder 42 in the dot memory while taking correspondence with the address of the semiconductor light emitting element 11.

  In the fluorescence amount discriminator 43, a fluorescence threshold TL2 is set. The fluorescence amount discriminator 43 receives an instruction signal from the timing controller 32 and discriminates whether or not each dot area addition value stored in the dot memory exceeds the fluorescence threshold value TL2. The fluorescence amount discriminator 43 outputs the dot area added value exceeding the fluorescence threshold TL2 to the calculation unit 50 together with the address of the semiconductor light emitting element 11 corresponding to the dot area added value.

  The calculation unit 50 in this embodiment includes a divider 51, a light detection monitor 52, and an A / D converter 53. The light detection monitor 52 detects the excitation light output from one semiconductor light emitting element 11 coupled to the optical fiber 13A through the optical fiber 13A, thereby detecting the excitation light supplied from the semiconductor light emitting element 11 to the sample S1. To detect. The light detection monitor 52 is, for example, a photomultiplier tube. The light detection monitor 52 outputs an electrical signal corresponding to the detected excitation light to the A / D converter 53. The A / D converter 53 converts an electrical signal that is an analog signal into excitation light amount data that is a digital signal, and outputs the data to the divider 51.

  The divider 51 receives the instruction signal from the timing controller 32, and the amount of fluorescence discriminated as the dot area addition value exceeds the fluorescence threshold TL2 by the fluorescence amount discriminator 43, and the sample S1 that has emitted the fluorescence. It calculates based on the light quantity of the excitation light supplied to. For this purpose, first, the divider 51 receives the dot area addition value output from the fluorescence amount discriminator 43 and the address of the semiconductor light emitting element 11 and the excitation light amount data output from the A / D converter 53. The divider 51 divides the dot area addition value by the excitation light amount data that is the light amount of the excitation light supplied to the sample S1, thereby calculating the corrected fluorescence amount that is the fluorescence light amount calculated based on the light amount of the excitation light. To do. The divider 51 outputs the calculated corrected fluorescence amount and the address of the semiconductor light emitting element 11 corresponding to each corrected fluorescence amount to the storage device 60. Here, the address of the semiconductor light-emitting element 11 corresponding to the corrected fluorescence amount refers to the address of the semiconductor light-emitting element 11 corresponding to the dot area addition value used when obtaining the corrected fluorescence amount.

  The storage device 60 receives as input the corrected fluorescence amount output from the divider 51 and the address of each semiconductor light emitting element 11 corresponding to each corrected fluorescence amount. The storage device 60 includes a memory, and stores the corrected fluorescence amount in correspondence with the address of the semiconductor light emitting element 11.

  After all of the corrected fluorescence amount output from the divider 51 is stored in the memory, the storage device 60 sends the address and end signal of the semiconductor light emitting element 11 stored in association with the corrected fluorescence amount to the timing controller 32. Output.

  In response to this, the timing controller 32 outputs an instruction signal to the LED power controller 12 so as to stop the supply of power to the semiconductor light emitting element 11 corresponding to the address output from the storage device 60. That is, the timing controller 32 sends an instruction signal to the LED power controller 12 to stop the supply of excitation light to the sample S1 that has emitted fluorescence whose light amount has been determined by the discriminator 40 to exceed the fluorescence threshold TL2. Output. In response to this instruction signal, the LED power controller 12 changes the power supplied to the semiconductor light emitting element 11 that supplies the excitation light to the corresponding sample S1, and stops the output of the excitation light.

  The timing controller 32 further outputs an instruction signal to the LED power controller 12 so as to increase the supply of power to the semiconductor light emitting element 11 having an address different from the address output from the storage device 60. That is, the timing controller 32 sends an instruction signal to the LED power controller 12 to increase the amount of excitation light to be supplied to the sample S1 that has emitted fluorescence for which the light amount is determined not to exceed the fluorescence threshold TL2 in the discrimination unit 40. Output. In response to this instruction signal, the LED power controller 12 changes the power supplied to the semiconductor light emitting element 11 that supplies the excitation light to the corresponding sample S1, thereby increasing the amount of excitation light. Thus, the timing controller 32 functions as excitation light control means.

  Next, a fluorescence measurement method using the fluorescence measurement device 1 will be described. First, the video signal threshold TL1 is set in the video signal level detector 31, and the fluorescence threshold TL2 is set in the fluorescence amount discriminator 43, respectively. The video signal threshold value TL1 is a threshold value that is set so that the video signal that captures fluorescence does not exceed the saturation level in the light detection unit 20. Therefore, the video signal threshold value TL1 is set to a value of 80 to 90% of the maximum video signal level.

  On the other hand, the fluorescence threshold value TL2 is a threshold value for capturing only dot area addition values that do not affect crosstalk even when a plurality of dot area addition values are simultaneously acquired. Therefore, the fluorescence threshold TL2 is set to a low value of about 1/4 to 1/8 of the maximum dot area addition value.

  After setting the video signal threshold value TL1 and the fluorescence threshold value TL2, in response to an instruction signal from the timing controller 32, the LED power controller 12 starts supplying power to the semiconductor light emitting element 11 while increasing the supply amount. Thereby, the excitation light emitted from the semiconductor light emitting element 11 is supplied to each of the plurality of samples S1 while increasing the amount of light (excitation light supply step). At this time, it is preferable to supply electric power so that at least the amount of increase in the amount of light of each semiconductor light emitting element 11 is maintained, and more preferably, the amount of light of each semiconductor light emitting element 11 is equal. Further, it is not always necessary to turn on all of the semiconductor light emitting elements 11, and only the region corresponding to the sample S1 may be turned on.

  The sample S1 is excited by the supplied excitation light and emits fluorescence. The emitted fluorescence is detected by the CCD camera 21 of the light detection unit 20 (light detection step). Fluorescence is detected by capturing a fluorescence image with the CCD camera 21 and converting it into an electrical signal. The electrical signal output from the CCD camera 21 is converted into a digital video signal by the A / D converter 23.

  The video signal is input to the video signal level detector 31. The video signal level detector 31 compares the input video signal with the video signal threshold value TL1. If the video signal exceeds the video signal threshold value TL1, the video signal level detector 31 sends a signal to the timing controller 32. In response to this signal, the timing controller 32 instructs the LED power controller 12 to stop increasing the supply power to the semiconductor light emitting element 11 and to keep the supply power constant in order to keep the amount of excitation light constant. Send.

  If the video signal exceeds the video signal threshold TL1, the video signal level detector 31 sends a signal to the timing controller 32 and simultaneously outputs the video signal to the image processor 41. The video signal output from the video signal level detector 31 is taken into the image processor 41 and stored in a frame memory included in the image processor 41, thereby generating image data.

  When the accumulation of the video signal is completed, a capture end signal is output from the image processor 41 to the timing controller 32. In response to this, image data is input from the image processor 41 to the dot area adder 42 from the timing controller 32, and the pixel data within the dot area of the image data is added to obtain the dot area addition value. An instruction signal to output to the fluorescence quantity discriminator 43 in correspondence with the address of the light emitting element 11 is output.

  In response to this instruction signal, the dot area adder 42 adds all the pixel data in the dot area of the image data. A method for adding pixel data in the dot area will be described in detail with reference to FIGS.

  FIG. 3A is a graph showing a part of the amount of fluorescence detected by the light detection unit 20 in correspondence with the arrangement of the sample. FIG. 3B is a graph showing the image data generated by the image processor 41. FIG. As shown to Fig.3 (a), the light quantity of the fluorescence generate | occur | produced for every sample S1 differs. As a result, as shown in FIG. 3B, in the dot area DA of the image data including each of the plurality of samples S1, the amount of signal included therein is different, and the shade representing the amount of fluorescence is different. In FIG. 3B, the dot area is shown darker as the amount of fluorescent light is larger.

  FIG. 4 is a diagram showing luminance signals on the horizontal video lines VL1 to VL8 crossing the dot area DA for one dot area DA displayed in an enlarged manner. A dot area addition value, which is a value obtained by adding all the pixel data in each dot area, is obtained by the dot area adder 42 as follows. That is, it is obtained by adding all the luminance signals in the dot area DA among the luminance signals on the horizontal video lines VL2 to VL7 crossing in one dot area DA. The dot area adder 42 obtains dot area addition values corresponding to the amounts of fluorescent light for all the dot areas DA. Since each dot area has a one-to-one correspondence with each of the plurality of samples S1, each dot area addition value is obtained corresponding to each semiconductor light emitting element 11 that supplies excitation light to each sample S1. Therefore, each dot area addition value is accompanied by the address of the corresponding semiconductor light emitting element 11.

  Returning to FIG. 1 again, the description of the fluorescence measurement method according to the present embodiment will be continued. The dot area addition value obtained by the dot area adder 42 is input to the fluorescence amount discriminator 43 in association with the address of the corresponding semiconductor light emitting element 11 and stored in the dot memory included in the fluorescence amount discriminator 43. . Thereafter, in the fluorescence amount discriminator 43, each dot area added value is compared with the fluorescence threshold TL2, and only the dot area added value exceeding the fluorescence threshold TL2 is output to the divider 51 (discrimination step).

  In addition to the dot area addition value described above, excitation light amount data that is the light amount of excitation light detected by the light detection monitor 52 is input from the A / D converter 53 to the divider 51. In response to the instruction signal from the timing controller 32, the divider 51 samples the sample S1 from which the fluorescence amount discriminated by the fluorescence amount discriminator 43 when the dot area addition value exceeds the fluorescence threshold value TL2 has emitted the fluorescence. Is calculated on the basis of the amount of excitation light supplied to the (calculation step). Specifically, the corrected fluorescence amount that is the fluorescence light amount calculated based on the light amount of the excitation light is specifically obtained by dividing the dot area addition value by the excitation light amount data in the divider 51.

  The corrected fluorescence amount thus obtained is input to the storage device 60 in association with the address of the corresponding semiconductor light emitting element 11 and stored in the memory included in the storage device 60. Thereafter, all the addresses of the semiconductor light emitting elements 11 corresponding to the dot area addition value determined to exceed the fluorescence threshold TL2 are output from the storage device 60 to the timing controller 32 together with the end signal.

  The timing controller 32 instructs the LED power controller 12 to stop the supply of the excitation light from the semiconductor light emitting element 11 corresponding to the dot area addition value determined to have exceeded the fluorescence threshold TL2 in the discrimination step to the sample S1. An instruction signal is output. The LED power controller 12 receives this instruction signal and stops supplying power to the corresponding semiconductor light emitting element 11. Thereby, the supply of the excitation light to the sample S1 that has emitted the distinguished fluorescence when the light amount exceeds the fluorescence threshold TL2 in the discrimination step is stopped (supply stop step).

  Furthermore, the timing controller 32 increases the supply of excitation light from the semiconductor light emitting element 11 corresponding to the dot area addition value determined as not exceeding the fluorescence threshold TL2 in the discrimination step to the sample S1. 12 outputs an instruction signal. In response to this instruction signal, the LED power controller 12 increases the supply of power to the semiconductor light emitting element 11 until the video signal indicating the amount of fluorescence detected by the light detection unit 20 exceeds the video signal threshold TL1. In this way, the light amount of the excitation light supplied to the sample S1 that has emitted the distinguished fluorescence when the light amount does not exceed the fluorescence threshold TL2 in the discrimination step is increased (excitation light amount increase step).

  Thereafter, a series of steps from the excitation light supply step to the excitation light amount increase step is repeated until the output of the LED power controller 12 becomes maximum. When the output of the LED power controller 12 reaches a maximum, an instruction signal is sent from the timing controller 32 to the fluorescence amount discriminator 43. In response to this instruction signal, the value of the fluorescence threshold value TL2 set in the fluorescence amount discriminator 43 is set to zero. Thereby, all the dot area addition values are input to the divider 51, and measurement is performed on the sample S1 that emits only weak fluorescence below the fluorescence threshold TL2. In this way, the corrected fluorescence amount is obtained for the fluorescence from all the samples S1. The corrected fluorescence amount thus determined is then input to the storage device 60 and stored in a memory included in the storage device 60. Thus, the measurement of the fluorescence emitted from the plurality of samples S1 is completed. Since the corrected fluorescence amount in the present embodiment is a relative value, it is compared with a table in which the actual fluorescence amount and the corrected fluorescence amount are associated in advance, or an equation that can be converted into the actual fluorescence amount is obtained. Alternatively, the actual fluorescence amount may be obtained by applying it.

  With reference to FIG. 5, a description will be given of measuring fluorescence emitted from a plurality of samples S <b> 1 by repeating a series of steps from the excitation light supply step to the excitation light amount increase step. FIG. 5 shows that the excitation light supply to the sample showing the dot area addition value (fluorescence amount) exceeding the fluorescence threshold TL2 is stopped, and the excitation light for the sample showing the dot area addition value (fluorescence amount) not exceeding the fluorescence threshold TL2 It is a figure for demonstrating typically the method to increase a light quantity and to obtain | require a dot area addition value (fluorescence amount) continuously.

  FIG. 5A shows dot area addition values in 12 dot areas DA1 to DA12. The alternate long and short dash line represents the fluorescence threshold value TL2, and the dot area addition value in the dot areas DA1, DA3, DA4, DA6, DA8, DA10, and DA12 exceeds the fluorescence threshold value TL2.

  FIG. 5B shows the case where the supply of the excitation light to the sample S1 corresponding to the dot areas DA1, DA3, DA4, DA6, DA8, DA10, DA12, which has shown the dot area addition value exceeding the fluorescence threshold TL2, is stopped. Indicates the dot area addition value. The dot area addition value in the dot area corresponding to the sample S1 for which the supply of excitation light is stopped indicates 0.

  FIG. 5C shows dot area addition values when the amount of excitation light supplied to the sample S1 corresponding to the dot areas DA2, DA5, DA7, DA9, and DA11 is increased. The alternate long and short dash line represents the fluorescence threshold TL2, and the dot area addition value in the dot areas DA2, DA5, DA9 exceeds the fluorescence threshold TL2.

  FIG. 5D shows dot area addition values when the supply of excitation light to the sample S1 corresponding to the dot areas DA2, DA5, and DA9, which has shown dot area addition values exceeding the fluorescence threshold TL2, is stopped. The dot area addition value in the dot area corresponding to the sample S1 for which the supply of excitation light is stopped indicates 0.

  FIG. 5E shows dot area addition values when the amount of excitation light supplied to the sample S1 corresponding to the dot areas DA7 and DA11 is increased. The alternate long and short dash line represents the fluorescence threshold TL2, and the dot area addition value in the dot areas DA7 and DA11 exceeds the fluorescence threshold TL2. Thereby, the dot area addition value of all the 12 dot areas DA1 to DA12, that is, the amount of fluorescence is obtained.

  Next, the effect of the fluorescence measuring apparatus and the fluorescence measuring method according to the present embodiment will be described. In the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, excitation light is supplied to each of the plurality of samples S1, and the amount of fluorescence emitted from each sample is supplied to each of the plurality of samples S1. Each is calculated based on the amount of light. Therefore, the fluorescence emitted from the plurality of samples S1 can be measured at the same time, and the time required for the measurement can be shortened.

  Further, in the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, the fluorescence amount discriminator 43 of the discrimination unit 40 discriminates whether or not the dot area addition value (fluorescence amount) exceeds the fluorescence threshold value TL2. Only for those exceeding the fluorescence threshold TL2, the divider 51 calculates the corrected fluorescence amount. Further, the supply of excitation light to the sample S1 whose dot area addition value (fluorescence amount) exceeds the fluorescence threshold TL2 is stopped, and continues only for the sample S1 whose dot area addition value does not exceed the fluorescence threshold TL2. The amount of fluorescence is measured. Therefore, when actually measuring a small amount of fluorescence, the supply of excitation light to a sample exhibiting a large amount of fluorescence is stopped. Therefore, in the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, it is possible to suppress the occurrence of crosstalk in which the fluorescence of one sample S1 affects the measurement region for another sample S1 in the vicinity. Thus, it is possible to acquire fluorescence amount data that is less affected by crosstalk.

  In particular, when the difference in the amount of fluorescence generated from the adjacent sample S1 is large (sample S1> sample S1 ′ in the fluorescence amount), accurate measurement of the sample S1 ′ showing a small fluorescence amount due to the influence of crosstalk. Becomes more difficult. This is because the fluorescence of the sample S1 may reach the detection region of the sample S1 ′ and be added to the original fluorescence amount of the sample S1 ′. However, in the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, when measuring a small amount of fluorescence, the supply of excitation light to a sample exhibiting a large amount of fluorescence is stopped. It is greatly suppressed.

  As described above, the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment can suppress the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples.

  Further, in the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, the dot area addition value (fluorescence amount) is increased by increasing the amount of excitation light supplied to the sample S1 that does not exceed the fluorescence threshold value TL2, and the dot area again. The added value (fluorescence amount) is measured. Therefore, when the excitation light is supplied with a uniform amount of light, even for a sample that can only emit weak fluorescence that is hidden by strong fluorescence, or for a sample that does not emit fluorescence with that excitation light amount, Measurement can be performed while suppressing the influence of crosstalk.

  Moreover, as described above, even weak fluorescence can be measured while suppressing the influence of crosstalk, the sensitivity width (dynamic range) that can be measured by the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment is significantly increased. In particular, in the detection of fluorescence by the CCD camera 21, the dynamic range is generally reduced because the capacity of each pixel of the CCD is small. However, in the fluorescence measurement device 1 and the fluorescence measurement method according to the present embodiment, even if a CCD camera is used for fluorescence detection, the measurement of relatively weak fluorescence is performed by increasing the supply amount of excitation light. Therefore, it is possible to increase the dynamic range.

  As described above, in the fluorescence measurement device 1 and the fluorescence measurement method according to this embodiment, the dynamic range can be increased without using a cooling CCD or the like. Therefore, it is possible to reduce the cost of the apparatus and the cost required for measurement.

  The timing controller 32 functioning as excitation light control means increases the supply amount of excitation light by changing the power supplied to each semiconductor light emitting element 11. For example, the supply amount of excitation light can be increased in this way.

  There are a plurality of semiconductor light emitting elements 11 serving as excitation light sources, which correspond to the plurality of samples S1 on a one-to-one basis. The timing controller 32 functioning as excitation light control means outputs an instruction signal to the LED power controller 12 so as to stop the output of excitation light from each semiconductor light emitting element 11, thereby exciting light for the sample S <b> 1. Supply will be stopped. For example, the supply of excitation light can be stopped in this way.

  Further, the plurality of samples S1 are two-dimensionally arranged. Even in such a case, the fluorescence measuring apparatus 1 can simultaneously measure fluorescence generated from a plurality of samples while suppressing the occurrence of crosstalk.

  In the fluorescence measuring apparatus 1, a semiconductor light emitting element 11 is used as an excitation light source. Therefore, the control of the excitation light quantity becomes easy, and in addition, the apparatus can be downsized and the power consumption can be reduced.

  In addition, the excitation light supply unit 10 includes an optical fiber 13 that functions as a light guide unit that guides the excitation light from the semiconductor light emitting element 11 to the plurality of samples S1. Therefore, excitation light can be easily supplied to each of the plurality of samples S1.

  The light detection unit 20 includes a CCD camera 21 that is a solid-state imaging device. Therefore, the fluorescence measuring apparatus 1 can easily measure fluorescence generated from a large number of samples at the same time.

  In the fluorescence measuring apparatus 1, correction is performed by dividing the dot area addition value (fluorescence amount) discriminated as exceeding the fluorescence threshold TL2 by the excitation light amount data (excitation light amount) supplied to the corresponding sample S1. The amount of fluorescence is calculated. Therefore, the fluorescence of samples with different amounts of supplied excitation light can be easily compared and examined by the corrected amount of fluorescence standardized with the amount of excitation light.

  In addition, in order to align the luminous efficiency of each semiconductor light emitting element 11 and the degree of increase when the light amount is increased more accurately, an initial correction process and an intermediate correction process may be performed.

  As an example of initial correction, there is correction by processing of acquired fluorescence data. For example, a standard sample is placed in all the wells formed on the plate P1, and the dot area adder 42 obtains a dot area addition value for the fluorescence generated from all the samples, and this is used as a calibration value. As a result, calibration values are obtained for all dot areas, and correction can be performed based on the calibration values.

  As another example of the initial correction, there is correction based on the light amount of the excitation light source. For example, a standard sample is put in all the wells formed on the plate P1, and the dot area adder 42 calculates the dot area addition value for the fluorescence generated from all the samples. The timing controller 32 instructs the LED power controller 12 to supply power to the semiconductor light emitting element 11 so that all the dot area addition values are the same. In response to this instruction, the power supplied to the semiconductor light emitting element 11 is adjusted.

  As an example of the intermediate correction, for example, excitation light emitted from one of the plurality of semiconductor light emitting elements 11 is detected and measured by the light detection monitor 52. Then, the correction is performed by using the value as the calibration value on the assumption that the value is the same as the excitation light emitted from the other semiconductor light emitting elements 11. The fluorescence measuring apparatus according to the present embodiment employs this intermediate correction method.

  Further, without performing intermediate correction, after performing initial correction, the amount of excitation light supplied from the semiconductor light emitting element 11 to the sample S1 is determined not by the supplied power but by the ON / OFF ratio of pulsed light oscillation. You may respond by changing. In this case, since all the semiconductor light emitting elements 11 change in the same amount of light, the same effect as the intermediate correction can be obtained.

(Second Embodiment)
With reference to FIG.6 and FIG.7, the structure of the fluorescence measuring apparatus which concerns on 2nd Embodiment is demonstrated. The fluorescence measurement apparatus according to the second embodiment does not stop the supply of excitation light by stopping the supply of power supplied to the excitation light source, but instead modulates the excitation light by modulating the intensity of the excitation light by the light modulation unit. Is different from the fluorescence measuring apparatus according to the first embodiment in that the supply of the light is stopped.

  FIG. 6 is a configuration diagram schematically showing a fluorescence measuring apparatus according to the second embodiment. As shown in FIG. 6, the fluorescence measurement device 70 according to the present embodiment includes an excitation light supply unit 80 that supplies excitation light to each of a plurality of samples, a light modulation unit 85 that modulates the excitation light, A light detection unit 90 that detects fluorescence emitted from a plurality of samples, a timing control unit 102 that controls excitation light supplied to the plurality of samples, and a discrimination unit that discriminates whether or not the amount of fluorescence exceeds a fluorescence threshold. 110 and a calculation unit 120 that calculates the corrected fluorescence amount.

  The excitation light supply unit 80 in this embodiment includes an excitation light source panel 81 and an excitation light source driving circuit 82. In addition, the light modulation unit 85 in the present embodiment includes a liquid crystal panel 86 and a liquid crystal panel drive circuit 87. FIG. 7 shows the relationship between the gel-like sample S <b> 2 whose fluorescence is measured by the fluorescence measuring device 1, the excitation light source panel 81, and the liquid crystal panel 86. The gel-like sample S2 is spread on one main surface of the plate P2 for measurement. The plate P2 is placed on the liquid crystal panel 86. The excitation light source panel 81 is driven by the excitation light source drive circuit 82 and supplies excitation light to the gel-like sample S2. The amount of excitation light emitted from the excitation light source panel 81 is changed by driving the excitation light source driving circuit 82.

  The liquid crystal panel 86 modulates the intensity of the excitation light by being turned on or off by the liquid crystal panel drive circuit 87. Specifically, the excitation light can be supplied to the corresponding region of the sample S2 by turning on the pixel, and the excitation light can be supplied to the region of the corresponding sample S2 by turning off the pixel. Can be canceled. Thus, the liquid crystal panel 86 and the liquid crystal panel drive circuit 87 function as the light modulator 85 that modulates the excitation light.

  In the present embodiment, each pixel of the liquid crystal panel 86 corresponds to the plurality of regions of the sample S2 on a one-to-one basis. Therefore, the excitation light supply unit 80 can supply excitation light to each of the plurality of regions of the sample S2 corresponding to the plurality of samples. In addition, you may make it respond | correspond one-to-one with the several area | region of sample S2 by making several pixels into 1 unit.

  Returning to FIG. 6 again, the description of the fluorescence measuring apparatus 70 will be continued. The light detection unit 90 detects fluorescence emitted from a plurality of regions of the sample S2. The light detection unit 90 in the present embodiment includes a CCD camera 91, a camera controller 92, and an A / D converter 93. An electrical signal obtained by detecting fluorescence with the CCD camera 91 is sent to the A / D converter 93 to generate a video signal. The A / D converter 93 outputs a video signal indicating the amount of fluorescence detected by the light detection unit 90 to the video signal level detector 101.

  In the video signal level detector 101, a video signal threshold TL1 is set. The video signal level detector 101 sends a signal to the timing controller 102 when the level of the video signal exceeds the video signal threshold value TL1.

  The timing controller 102 receives a signal from the video signal level detector 101, and instructs the excitation light source driving circuit 82 about the power supplied to the excitation light source panel 81 (for example, an instruction signal for making the supply power constant, etc.) ) The timing controller 102 functions as excitation light control means as will be described later, and in the present embodiment, outputs from the respective parts constituting the fluorescence measuring apparatus 1 are input, and instructions are given to other parts according to the input values. It also functions as a control means for outputting a signal.

  The discriminating unit 110 in the present embodiment includes an image processor 111 and a video signal discriminator 112. The image processor 111 includes a frame memory that takes in and stores the video signal output from the video signal level detector 101 as an input. The frame memory of the image processor 111 stores the accumulated video signal as image data. The image processor 111 outputs the image data stored in the frame memory to the video signal discriminator 112. Further, the image processor 111 sends a capture end signal to the timing controller 102 when the capture of the video signal from the video signal level detector 101 is completed.

  In the video signal discriminator 112, a fluorescence threshold TL2 is set. The video signal discriminator 112 receives an instruction signal from the timing controller 102, and discriminates whether data corresponding to each pixel of the liquid crystal panel 86 in the image data (hereinafter simply referred to as pixel data) exceeds the fluorescence threshold value TL2. To do. The video signal discriminator 112 outputs pixel data exceeding the fluorescence threshold TL2 to the arithmetic unit 120 together with the pixel address of the CCD camera 91 corresponding to the pixel data. Here, the pixel address of the CCD camera is a pointer that specifies the position of the pixel arranged in a two-dimensional array in the CCD camera 21 by, for example, the number of columns and the number of rows. The pixel data is not limited to data corresponding to each pixel of the liquid crystal panel, and may be data corresponding to a desired unit area in the image data. Therefore, for example, data corresponding to a plurality of pixels of the liquid crystal panel may be used.

  In the present embodiment, the number of pixels of the liquid crystal panel 86 and the number of pixels of the CCD camera 91 are the same. Further, each pixel of the liquid crystal panel 86 and each pixel of the CCD camera 91 are optically equivalent to the relationship between the object plane and the imaging plane. Accordingly, each pixel of the liquid crystal panel 86 and each pixel of the CCD camera 91 have a one-to-one correspondence. In the present embodiment, each pixel of the liquid crystal panel 86 and each region of the sample S2 have a one-to-one correspondence. Therefore, each region of the sample S2 and each pixel of the CCD camera 91 has a one-to-one relationship. It can be said that it corresponds. However, the number of each element unit (each pixel in the liquid crystal panel 86, each pixel in the CCD camera 91, each region in the sample S2) is not necessarily the same, and at least more than the number necessary to correspond to the sample S2. It ’s fine. Further, a plurality of pixels and pixels may correspond to a desired unit region of the sample S2.

  The arithmetic unit 120 in this embodiment includes a divider 121, a light detection monitor 122, and an A / D converter 123. Excitation light supplied from the excitation light source panel 81 to each region of the sample S2 is guided to the light detection monitor 122 by an optical system including, for example, a mirror and detected by the light detection monitor 122. The light detection monitor 122 outputs an electrical signal corresponding to the detected excitation light to the A / D converter 123. The A / D converter 123 converts the electrical signal that is an analog signal into excitation light amount data that is a digital signal, and outputs the data to the divider 121.

  The divider 121 receives the instruction signal from the timing controller 102, and determines the amount of fluorescence discriminated when the pixel data exceeds the fluorescence threshold TL2 by the video signal discriminator 112 as the region of the sample S2 that has emitted the fluorescence. It calculates based on the light quantity of the excitation light supplied to. For this purpose, first, the divider 121 receives the pixel data output from the video signal discriminator 112, the pixel address of the CCD camera 91, and the excitation light amount data output from the A / D converter 123. The divider 121 divides the pixel data by the excitation light amount data that is the light amount of the excitation light supplied to the corresponding region of the sample S2, thereby correcting the fluorescence amount that is the fluorescence light amount calculated based on the light amount of the excitation light. Is calculated. The divider 121 outputs the calculated corrected fluorescence amount and the pixel address of the CCD camera 91 corresponding to each corrected fluorescence amount. Here, the address of the pixel of the CCD camera 91 corresponding to each corrected fluorescence amount refers to the address of the pixel of the CCD camera 91 corresponding to the pixel data used when obtaining each corrected fluorescence amount.

  The storage device 130 receives the corrected fluorescence amount output from the divider 121 and the pixel address of the CCD camera 91 corresponding to each corrected fluorescence amount. The storage device 130 includes a memory, and the corrected fluorescence amount is stored in this memory in association with the pixel address of the CCD camera 91.

  After all the corrected fluorescence amount output from the divider 121 is stored in the memory, the storage device 130 outputs the end signal and the address of the pixel of the CCD camera 91 stored in association with the corrected fluorescence amount to the timing controller 102. To do.

  In response to this, the timing controller 102 suspends the supply of the excitation light by modulating the intensity of the excitation light in the pixel of the liquid crystal panel 86 corresponding to the pixel address of the CCD camera 91 output from the storage device 130. An instruction signal is output to the liquid crystal panel drive circuit 87. That is, the timing controller 102 outputs an output from the storage device 130 in order to stop supplying excitation light to the corresponding region of the sample S2 that has emitted the fluorescence that has been determined by the discriminator 110 that the light amount exceeds the fluorescence threshold TL2. An instruction signal for turning off the pixels of the liquid crystal panel 86 corresponding to the pixel address of the CCD camera 91 is output to the liquid crystal panel drive circuit 87. In response to this instruction signal, the liquid crystal panel drive circuit 87 turns off the pixels of the corresponding liquid crystal panel 86 and stops the supply of excitation light to the corresponding region of the sample S2. As described above, each pixel of the liquid crystal panel 86 and each pixel of the CCD camera 91 have a one-to-one correspondence. Therefore, by specifying the address of the pixel of the CCD camera 91, the corresponding pixel of the liquid crystal panel 86 is changed. Can be specified.

  Further, the timing controller 102 outputs an instruction signal to the excitation light source driving circuit 82 so as to change, that is, increase the power supplied to the excitation light source panel 81 in order to increase the amount of excitation light. Since the pixel of the liquid crystal panel 86 corresponding to the pixel address of the CCD camera 91 output from the storage device 130 is OFF, it corresponds to an address different from the pixel address of the CCD camera 91 output from the storage device 130. The excitation light supplied through the pixels of the liquid crystal panel 86 is increased and supplied to the sample S2. As a result, the light amount of the excitation light supplied to the region of the sample S2 that has emitted the fluorescence determined by the discriminator 110 that the light amount does not exceed the fluorescence threshold TL2 is increased. Thus, the liquid crystal panel 86 and the liquid crystal panel drive circuit 87 function as the light modulator 85, and the timing controller 102 functions as the excitation light control means.

  Next, a fluorescence measurement method using the fluorescence measurement device 70 will be described. First, the video signal threshold TL1 is set in the video signal level detector 101, and the fluorescence threshold TL2 is set in the video signal discriminator 112, respectively. The video signal threshold value TL1 is a threshold value that is set so that the video signal that captures fluorescence does not exceed the saturation level in the light detection unit 20. Therefore, the video signal threshold value TL1 is set to a value of 80 to 90% of the maximum video signal level.

  On the other hand, the fluorescence threshold value TL2 is a threshold value set to guarantee a minimum level as data that does not impair the S / N ratio for the video signal to be captured. Therefore, the fluorescence threshold TL2 is set to a low value of about 1/4 to 1/8 of the maximum video signal.

  After setting the video signal threshold value TL1 and the fluorescence threshold value TL2, upon receiving an instruction signal from the timing controller 102, the excitation light source driving circuit 82 starts to supply power to the excitation light source panel 81 while increasing the supply amount. Further, all the pixels of the liquid crystal panel 86 are turned on by the liquid crystal panel drive circuit 87. Thereby, the excitation light emitted from the excitation light source panel 81 is supplied to each of the plurality of regions of the sample S2, which is a plurality of samples, while increasing the amount of light (excitation light supply step). It is not always necessary to turn on all the pixels of the liquid crystal panel 86, and only the region corresponding to the sample S2 may be turned on.

  The sample S2 emits fluorescence according to the supplied excitation light. The emitted fluorescence is detected by the CCD camera 91 of the light detection unit 90 (light detection step). The fluorescence is captured by the CCD camera 91 and then converted into a digital video signal by the A / D converter 93.

  The video signal is input to the video signal level detector 101. The video signal level detector 101 compares the input video signal with the video signal threshold value TL1. If the video signal exceeds the video signal threshold TL1, the video signal level detector 101 sends a signal to the timing controller 102. In response to this signal, the timing controller 102 instructs the excitation light source drive circuit 82 to stop increasing the supply power to the excitation light source panel 81 and to keep the supply power constant in order to keep the amount of excitation light constant. Send a signal.

  If the video signal exceeds the video signal threshold TL1, the video signal level detector 101 sends a signal to the timing controller 102 and simultaneously outputs the video signal to the image processor 111. The output video signal is captured by the image processor 111 and stored in a frame memory included in the image processor 111, thereby generating image data. FIG. 8 is a diagram schematically showing image data generated by the image processor 111.

  When the accumulation of the video signal is completed, a capture end signal is output from the image processor 111 to the timing controller 102. In response to this, image data is input from the image processor 111 to the video signal discriminator 112 from the timing controller 102, the data of each pixel of the image data is compared with the fluorescence threshold value TL2, and the fluorescence threshold value is compared. An instruction signal indicating that only pixel data larger than TL2 is output to the divider 121 is output.

  In response to this instruction signal, the video signal discriminator 112 discriminates whether or not the pixel data exceeds the fluorescence threshold value TL2, and outputs only the pixel data exceeding the fluorescence threshold value TL2 to the divider 121 (discrimination step). .

  In addition to the pixel data described above, excitation light amount data that is the amount of excitation light detected by the light detection monitor 122 is input from the A / D converter 123 to the divider 121. In response to the instruction signal from the timing controller 102, the divider 121 determines the region of the sample S2 in which the amount of fluorescence discriminated when the pixel data exceeds the fluorescence threshold TL2 by the video signal discriminator 112. Is calculated on the basis of the amount of excitation light supplied to the (calculation step). Thus, the corrected fluorescence amount, which is the fluorescence light amount calculated based on the light amount of the excitation light, is specifically obtained by dividing the pixel data by the excitation light amount data.

  The corrected fluorescence amount thus obtained is input to the storage device 130 in association with the pixel address of the corresponding CCD camera 91 and stored in the memory included in the storage device 130. Thereafter, all the addresses of the pixels of the CCD camera 91 corresponding to the pixel data determined to exceed the fluorescence threshold TL2 are output from the storage device 130 to the timing controller 102 together with the end signal.

  The timing controller 102 turns off the pixels of the liquid crystal panel 86 corresponding to the pixel data discriminated as exceeding the fluorescence threshold TL2 in the discrimination step, and stops supplying the excitation light to the corresponding region of the sample S2. Then, an instruction signal is output to the liquid crystal panel drive circuit 87. The liquid crystal panel drive circuit 87 receives this instruction signal and turns off the corresponding pixel of the liquid crystal panel 86. Accordingly, the supply of excitation light to the region of the sample S2 that has emitted the distinguished fluorescence when the light quantity exceeds the fluorescence threshold TL2 in the discrimination step is stopped (supply stop step).

  Further, the timing controller 102 increases the amount of excitation light supplied to the region of the sample S2 corresponding to the pixel data identified as not exceeding the fluorescence threshold value TL2 in the discrimination step. An instruction signal is output. In response to this instruction signal, the excitation light source drive circuit 82 increases the output of the excitation light source panel 81 until the video signal indicating the amount of fluorescent light detected by the light detection unit 90 exceeds the video signal threshold TL1. Since the pixel of the liquid crystal panel 86 corresponding to the region of the sample S2 that emits the fluorescence that has been distinguished as having exceeded the fluorescence threshold TL2, the pixel is discriminated that the light quantity does not exceed the fluorescence threshold TL2 in the discrimination step. The amount of excitation light supplied to the region of the sample S2 that has emitted the fluorescent light is increased (excitation light amount increasing step).

  Thereafter, a series of steps from the excitation light supply step to the excitation light amount increase step is repeated until the output of the excitation light source driving circuit 82 becomes maximum. When the output of the excitation light source driving circuit 82 reaches the maximum, an instruction signal is sent from the timing controller 102 to the video signal discriminator 112. In response to this instruction signal, the value of the fluorescence threshold TL2 set in the video signal discriminator 112 is set to zero. As a result, all pixel data is input to the divider 121, and measurement is performed on the sample S2 that emits only weak fluorescence below the fluorescence threshold TL2. In this way, the corrected fluorescence amount is obtained for the fluorescence from all regions of the sample S2. The corrected fluorescence amount thus determined is then input to the storage device 130 and stored in a memory included in the storage device 130. Thus, measurement of fluorescence emitted from the plurality of regions of the sample S2 is completed. Since the corrected fluorescence amount in the present embodiment is a relative value, it is compared with a table in which the actual fluorescence amount and the corrected fluorescence amount are associated in advance, or an equation that can be converted into the actual fluorescence amount is obtained. Alternatively, the actual fluorescence amount may be obtained by applying it.

  With reference to FIG. 9, it will be described that fluorescence emitted from a plurality of regions of the sample S2 is measured by repeating a series of steps from the excitation light supply step to the excitation light amount increase step. FIG. 9 shows that the supply of excitation light to the sample region showing the fluorescence amount exceeding the fluorescence threshold TL2 is stopped, the excitation light amount for the sample region showing the fluorescence amount not exceeding the fluorescence threshold TL2 is increased, and the fluorescence continues. It is a figure which illustrates typically the method of calculating | requiring quantity with the video signal detected by the photon detection part 90. FIG.

  FIG. 9A shows a video signal of a portion corresponding to the line I-I ′ in FIG. 8. The alternate long and short dash line represents the fluorescence threshold TL2. Video signals in the minute portions R2 and R4 exceed the fluorescence threshold TL2. The video signals in the minute portions R1, R3, R5 do not exceed the fluorescence threshold TL2.

  FIG. 9B shows a video signal when the supply of excitation light to the region of the sample S2 corresponding to the minute portions R2 and R4 that have shown the video signal exceeding the fluorescence threshold TL2 is stopped. The video signal in the minute portion corresponding to the region of the sample S2 where the supply of the excitation light is stopped indicates 0.

  FIG. 9C shows a video signal when the amount of excitation light supplied to the region of the sample S2 corresponding to the minute portions R1, R3, and R5 is increased. The alternate long and short dash line represents the fluorescence threshold TL2. The video signals in the minute portions R12, R31, R33, and R51 exceed the fluorescence threshold value TL2. The video signals in the minute portions R11, R32, R52 do not exceed the fluorescence threshold TL2.

  FIG. 9D shows a video signal when supply of excitation light to the region of the sample S2 corresponding to the minute portions R12, R31, R33, and R51 that has shown the video signal exceeding the fluorescence threshold TL2 is stopped. The video signal in the minute portion corresponding to the region of the sample S2 where the supply of the excitation light is stopped indicates 0.

  FIG. 9E shows a video signal when the amount of excitation light supplied to the region of the sample S2 corresponding to the minute portions R11, R32, and R52 is increased. The alternate long and short dash line represents the fluorescence threshold TL2. The video signals in the minute portions R112, R32, and R521 exceed the fluorescence threshold TL2. The video signals in the minute portions R111 and R522 do not exceed the fluorescence threshold value TL2.

  Next, the effect of the fluorescence measuring apparatus and the fluorescence measuring method according to the present embodiment will be described. In the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment, excitation light is supplied to each of the plurality of regions of the sample S2, and the amount of fluorescence emitted from each region is supplied to each of the plurality of regions. Each is calculated based on the amount of light. Therefore, the fluorescence emitted from a plurality of regions of the sample S2 can be measured simultaneously, and the time required for the measurement can be shortened.

  Further, in the fluorescence measurement device 70 and the fluorescence measurement method according to this embodiment, the divider 121 calculates the corrected fluorescence amount only for pixel data (fluorescence amount) exceeding the fluorescence threshold TL2. Further, after the supply of excitation light to the region of the sample S2 whose pixel data exceeds the fluorescence threshold TL2, the amount of fluorescence continues for the region of the sample S2 whose pixel data does not exceed the fluorescence threshold TL2. Measured. Therefore, when actually measuring a small amount of fluorescence, the supply of excitation light to the region of the sample exhibiting a large amount of fluorescence is stopped. Therefore, in the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment, it is possible to suppress the occurrence of crosstalk, and it is possible to acquire fluorescence amount data that is less affected by crosstalk.

  In particular, even when the difference in the amount of fluorescence generated from the adjacent region of the sample S2 is large, the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment have a large fluorescence amount when measuring a small fluorescence amount. Since the supply of excitation light to the region of the sample indicating is stopped, the occurrence of problems due to crosstalk is greatly suppressed.

  As described above, the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment can suppress the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples.

  Further, in the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment, the amount of excitation light is increased with respect to the region of the sample S2 where the pixel data (fluorescence amount) does not exceed the fluorescence threshold TL2, and then fluorescence is again performed. Is measuring. Therefore, even for samples that can only emit weak fluorescence that is hidden by strong fluorescence, or for samples that do not emit fluorescence with the amount of excitation light before the increase, measure the amount of fluorescence while suppressing the influence of crosstalk. It becomes possible.

  Moreover, as described above, even weak fluorescence can be measured while suppressing the influence of crosstalk, so the sensitivity width (dynamic range) that can be measured by the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment is increased. As described above, in the fluorescence measurement device 70 and the fluorescence measurement method according to the present embodiment, the dynamic range can be increased without using a cooling CCD or the like. Therefore, the cost of the apparatus can be reduced.

  In the fluorescence measuring apparatus 70, each pixel of the liquid crystal panel 86 is turned on or off by the liquid crystal panel drive circuit 87 to modulate the intensity of the excitation light. That is, the excitation light is supplied to the region of the sample S2 corresponding to the pixel of the liquid crystal panel 86 that is turned on, and the excitation light is not supplied to the region of the sample S2 that corresponds to the pixel that is turned off. For example, as described above, the supply of excitation light is stopped by the liquid crystal panel 86 and the liquid crystal panel drive circuit 87 that function as the light modulation unit.

  The light detection unit 90 includes a CCD camera 91 that is a solid-state imaging device. Therefore, the fluorescence measuring apparatus 70 can easily measure fluorescence generated from a large number of samples simultaneously.

  In the fluorescence measuring device 70, correction is performed by dividing pixel data (fluorescence amount) discriminated as exceeding the fluorescence threshold TL2 by excitation light amount data (excitation light amount) supplied to the corresponding region of the sample S2. The amount of fluorescence is calculated. Therefore, the fluorescence of samples with different amounts of supplied excitation light can be easily compared and examined by the corrected amount of fluorescence standardized with the amount of excitation light.

  The light modulation unit is not limited to the liquid crystal panel, and may include, for example, a DMD (Digital Micromirror Device) or a simple shutter structure. Moreover, you may increase the light quantity of excitation light by modulating the intensity | strength of excitation light in a light modulation part. In this case, the output of the excitation light source may be constant, or finer control may be performed by adjusting the output intensity of the excitation light source.

  The preferred embodiments of the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the solid-state imaging device included in the light detection unit is not limited to a CCD camera, and may be a MOS or a photomultiplier tube having a plurality of detection areas. The plurality of samples may not be arranged in a two-dimensional array.

  Alternatively, the excitation light amount (semiconductor light emitting element, excitation light source panel, etc.) may be lit in pulses and the duty ratio may be changed to increase the excitation light amount. Further, the amount by which the excitation light amount is increased is not limited to the method of determining with reference to the video signal threshold value TL1, but may be dealt with by increasing the output of the excitation light source by a preset multiple, for example.

  The calculation of the corrected fluorescence amount in the calculation units 50 and 120 may be performed by a method other than dividing the data representing the fluorescence amount (for example, dot area addition value, pixel data, etc.) by the data representing the excitation light amount. For example, a standard sample whose data is known in advance may be measured simultaneously and compared with the data. Further, the calculation of the corrected fluorescence amount may not be performed by the calculation units 50 and 120. For example, the calculation units 50 and 120 may output the excitation light amount and the fluorescence amount, and the corrected fluorescence amount may be calculated by another computer or the like. In addition, the calculation of the corrected fluorescence amount is not performed every time whether or not the fluorescence amount exceeds the fluorescence threshold value TL2, for example, after acquiring and detecting the fluorescence amount for a part or all of the samples, The corrected fluorescence amount may be calculated collectively for some or all of the samples. Alternatively, for example, correction fluorescence amount calculation processing may be performed in parallel with detection of fluorescence from another sample.

  In addition to blocking the excitation light irradiation for the sample (or sample region) for which measurement has been completed, the acquisition of data on the detection side corresponding to the sample (or sample region) is also stopped. May be. In this case, the data acquisition may be stopped not only by the control by the detector but also by the control by the data processing unit.

  INDUSTRIAL APPLICABILITY The present invention can be used as a fluorescence measuring apparatus and a fluorescence measuring method that can suppress the occurrence of crosstalk when simultaneously measuring fluorescence generated from a plurality of samples.

It is a lineblock diagram showing roughly about a fluorescence measuring device concerning a 1st embodiment. It is a figure for demonstrating the relationship between a some sample, a semiconductor light-emitting device, and an optical fiber. (A) The graph which shows the fluorescence amount detected by the light detection part, (b) Image data showing the fluorescence amount of the sample. It is a figure showing the luminance signal on the horizontal video line which crosses the dot area displayed by expanding. It is a figure for demonstrating the method to measure the fluorescence discharge | released from a some sample. It is a lineblock diagram showing roughly about a fluorescence measuring device concerning a 2nd embodiment. It is a figure for demonstrating the relationship between the sample on a plate, an excitation light source panel, and a liquid crystal panel. This is image data generated by the frame memory. It is a figure for demonstrating the method to measure the fluorescence discharge | released from the several area | region of a sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,70 ... Fluorescence measuring apparatus 10, 80 ... Excitation light supply part, 11 ... Semiconductor light emitting element, 12 ... LED power controller, 13, 13A ... Optical fiber, 20, 90 ... Light detection part, 21, 91 ... CCD camera , 22, 92 ... camera controller, 23, 93 ... A / D converter, 31, 101 ... video signal level detector, 32, 102 ... timing controller, 40, 110 ... discriminator, 41, 111 ... image processor , 42 ... Dot area adder, 43 ... Fluorescence quantity discriminator, 50, 120 ... Calculation unit, 51, 121 ... Divider, 52, 122 ... Photodetection monitor, 53, 123 ... A / D converter, 60, 130 DESCRIPTION OF SYMBOLS ... Memory | storage device, 81 ... Excitation light source panel, 82 ... Excitation light source drive circuit, 85 ... Light modulation part, 86 ... Liquid crystal panel, 87 ... Liquid crystal panel drive circuit, 112 ... Video signal discriminator, S1, S ... sample, P1, P2 ... plate

Claims (11)

An excitation light supply unit that has an excitation light source and supplies excitation light output from the excitation light source to each of a plurality of samples;
Light detection means for detecting fluorescence emitted from the plurality of samples;
Discrimination means for discriminating whether or not the amount of fluorescence emitted from each of the plurality of samples and detected by the light detection means exceeds a predetermined fluorescence threshold;
An arithmetic means for performing processing for calculating the light quantity of the fluorescence discriminated as having exceeded the fluorescence threshold by the discrimination means based on the light quantity of the excitation light supplied to the sample that has emitted the fluorescence;
Discontinuing the supply of the excitation light to the sample that has emitted the distinguished fluorescence when the light quantity exceeds the fluorescence threshold in the discrimination means, and discriminating that the light quantity does not exceed the fluorescence threshold in the discrimination means And an excitation light control means for increasing the amount of the excitation light supplied to the sample that has emitted the emitted fluorescence.   The fluorescence measurement apparatus according to claim 1, wherein the excitation light control unit increases a light amount of the excitation light by changing electric power supplied to the excitation light source. The excitation light source is a plurality, and at least one-to-one correspondence with the plurality of samples,
The pumping light control unit changes the power supplied to the pumping light source, and stops the pumping light supply by stopping the output of the pumping light from the pumping light source. Or the fluorescence measuring apparatus of Claim 2.   A light modulating unit that modulates the pumping light output from the pumping light source; and modulating the intensity of the pumping light in the light modulating unit to increase the amount of the pumping light. The fluorescence measuring device according to any one of claims 1 to 3, wherein   It further comprises light modulation means for modulating the excitation light output from the excitation light source, and by modulating the intensity of the excitation light in the light modulation means, the excitation light to the plurality of samples is modulated. Supply is stopped, The fluorescence measuring apparatus as described in any one of Claims 1-4 characterized by the above-mentioned.   The fluorescence measuring apparatus according to any one of claims 1 to 5, wherein the plurality of samples are arranged in a two-dimensional manner.   The fluorescence measurement apparatus according to claim 1, wherein the excitation light source is a semiconductor light emitting element.   The fluorescence measurement apparatus according to claim 1, wherein the excitation light supply unit further includes a light guide that guides the excitation light from the excitation light source to each of the plurality of samples. .   The fluorescence measurement apparatus according to claim 1, wherein the light detection unit is a solid-state imaging device.   The arithmetic means divides the light quantity of the fluorescence discriminated as having exceeded the fluorescence threshold by the discrimination means by the light quantity of the excitation light supplied to the sample that has emitted the fluorescence, The fluorescence measurement apparatus according to claim 1, wherein the fluorescence light amount is calculated. An excitation light supply step of supplying excitation light output from the excitation light source to each of a plurality of samples;
A light detection step of detecting fluorescence emitted from the plurality of samples;
A discrimination step for discriminating whether or not the amount of fluorescence emitted from each of the plurality of samples and detected in the light detection step exceeds a predetermined fluorescence threshold;
A calculation step for performing a process for calculating the amount of fluorescence discriminated as having exceeded the fluorescence threshold in the discrimination step based on the amount of excitation light supplied to the sample that has emitted the fluorescence;
A supply stopping step of stopping the supply of the excitation light to the sample that has emitted the fluorescence that has been discriminated when the light quantity exceeds the fluorescence threshold in the discrimination step;
A fluorescence measurement method comprising: an excitation light quantity increasing step for increasing the light quantity of the excitation light supplied to the sample that has emitted the fluorescence that has been discriminated if the light quantity does not exceed the fluorescence threshold value in the discrimination step.

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