JP2004279258A - Light measurement apparatus and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a light measurement apparatus and a method capable of always, reliably and accurately measuring a light in an optimal state. <P>SOLUTION: As an optical transmittance of a ND (nutral density) filter 17 is changed based on a comparison result of an output from a photo multiplier 13 in response to fluorescence intensity from a spot (a probe) of a micro array chip 7 and a reference, the output from the photo multiplier 13 is always and regularly controlled and corrected by a fluorescence intensity conversion process based on information on the optical transmittance of the ND filter 17. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
INDUSTRIAL APPLICABILITY The present invention is used for testing biologically related substances such as genes, for example, reading a microarray chip used for DNA analysis, epidemiological analysis, and the like, and specifically, a light measuring device and method for measuring fluorescence from the microarray chip. It is about.
[0002]
[Prior art]
In recent years, the development of technology in the field of genetic engineering has been remarkable, and, for example, research has been advanced to decode the base sequence of the human genome, which is considered to reach 100,000.
[0003]
On the other hand, microarray technology has been used for studies such as enzyme immunoassays and fluorescent antibody methods which are used for various diagnoses and utilize antigen-antibody reactions, and for searching for DNA that affects various genetic diseases.
[0004]
The microarray technology is a technology that uses, for example, a microarray chip in which cDNA or oligo DNA is spotted in a matrix at high density (interval of several hundred μm or less) as a probe on a Si wafer, a slide glass, or a membrane filter.
[0005]
Such a microarray technique is, for example, using a pipette to remove DNA extracted from cells of healthy subjects labeled with a fluorescent dye or DNA extracted from cells of a sample having a genetic disease labeled with a fluorescent dye using a pipette. Of each probe. Then, the DNA of each specimen and the probe are hybridized, and in this state, each probe is irradiated with excitation light for exciting each fluorescent dye, and the fluorescence emitted from the probe is detected by a photodetector. Thereafter, from the results of the fluorescence detection on the microarray chip, the DNA of each sample was determined to be hybridized with which probe, and by comparing the hybridized DNA, the DNA expressed or deficient due to the disease was determined. It is designed to identify DNA.
[0006]
By the way, as a technique for detecting fluorescence emitted from each probe of a microarray chip used in the microarray technique, for example, a technique disclosed in Patent Document 1 is known.
[0007]
FIG. 17 shows a schematic configuration of a microarray chip reader disclosed in Patent Document 1. As shown in FIG. In this case, 101 is a microarray chip, 102 is a slide glass, 103 is a combined object (object to be detected), 104 and 105 are step motors, 106 is a stage, 107 is an excitation light source, 108 is a collimator lens, 109 is a deflecting beam splitter, 110 is a condenser lens, 111 is a photomultiplier, and 112 is position indicating means.
[0008]
In such a configuration, first, the microarray chip 101 is set at a predetermined position on the stage 106. At this time, each position where the probe on the microarray chip 101 was spotted is associated with a position on the stage 106 in the X-axis direction and a position in the Y-axis direction. This association is input from the position indicating means 112 to each of the step motors 104 and 105. On the other hand, the excitation light source 107 emits the excitation light L. This excitation light L is incident on the collimator lens 108 and is converted into a parallel light beam. The excitation light L converted into a parallel light beam passes through the deflecting beam splitter 109, and is condensed on the microarray chip 101 provided on the stage 106 by the condensing lens 110.
[0009]
Each of the step motors 104 and 105 drives the stage 106 in the XY plane so that the excitation light L irradiates the first predetermined position on the microarray chip 101 based on the designated position input from the position designating means 112. Stop at position. The excitation light L irradiates the first predetermined position on the microarray chip 101 with a spot diameter wider than that of the combined substance 103. When the combined substance 103 is present at the first predetermined position irradiated with the excitation light L, The combined substance 103 is entirely irradiated with the excitation light L to excite the fluorescent dye of the combined substance 103 and emit fluorescence k. When the binding substance 103 does not exist at the first predetermined position irradiated with the excitation light L, the fluorescence k does not emit from the microarray chip 101. When the fluorescent substance k is emitted when the compound 103 is present, the fluorescent light k passes through the condensing lens 110 and the deflecting beam splitter 109 in sequence, enters the photomultiplier 111, and receives an electric signal corresponding to the amount of light. And output to an external processing device. The information on the predetermined position irradiated with the excitation light L is input from the position indicating means 112 to the external output device, and the predetermined position is associated with the presence or absence of the detection of the fluorescence k and the light amount.
[0010]
Patent Document 2 discloses a photomultiplier in which a microarray chip is irradiated with stable and stable illumination light by detecting a light amount from a light source and controlling an output light amount of the light source based on the detected value. Discloses measuring the fluorescence intensity of each spot.
[0011]
[Patent Document 1]
JP 2000-131237 A
[0012]
[Patent Document 2]
U.S. Pat. No. 6,406,849
[0013]
[Patent Document 3]
JP-A-8-334709
[0014]
[Patent Document 4]
JP-A-5-183851
[0015]
[Problems to be solved by the invention]
The apparatus disclosed in Patent Document 1 photoelectrically converts the amount of the fluorescent light k emitted from the microarray chip 101 using the photomultiplier 111, and digitizes the strength of the output signal using a data processing unit or the like.
[0016]
By the way, the light receiving element such as the photomultiplier 111 has a predetermined intensity range (dynamic range) of light capable of photoelectrically converting incident light. Therefore, the amount of fluorescent light emitted by hybridization in the microarray chip 101 is reduced. There are restrictions on the measurable range.
[0017]
However, the hybridization between the probe cDNA on the microarray chip 101 and the DNA labeled with the fluorescent dye varies depending on the variation during the reaction, that is, depending on the degree of expression, the case where extremely bright fluorescence is emitted under a single illumination condition. It may emit weak fluorescence.
[0018]
Therefore, when the photomultiplier 111 is used, the dynamic range may determine the dynamic range of the detection system. This is because if the driving voltage (HV: also referred to as high volt) of the photomultiplier 111 is adjusted so that the amount of fluorescence that emits the weakest fluorescence at the spot (probe 103) of the microarray chip 101 can be measured, When the amount of fluorescence from the spot (probe 103) that emits the brightest fluorescence is measured, the output of the photomultiplier 111 overflows, and the accurate amount of fluorescence cannot be measured.
[0019]
Therefore, it is conceivable to obtain an output signal while changing the drive voltage of the photomultiplier 111 for each amount of fluorescence emitted from each spot (probe 103). However, since the S / N changes depending on the drive voltage, the subsequent signal is changed. It is not possible to obtain an accurate output suitable for comparing the amount of fluorescence of each spot.
[0020]
The photomultiplier 111 has the best quantum efficiency depending on the drive voltage in terms of characteristics, and it is desirable to use the photomultiplier 111 with the drive voltage in the state with the best quantum efficiency. However, if the driving voltage is changed as described above, the performance of the photomultiplier 111 cannot be sufficiently exhibited.
[0021]
Further, instead of the photomultiplier 111, a CCD camera capable of two-dimensional photometry may be used. If such a CCD camera is used, fluorescence from all the spots (probes 103) of the microarray chip 101 can be captured by one exposure in one exposure.
[0022]
However, since the dynamic range of such a CCD camera is determined, if the dynamic range is narrow, spots with a weak amount of fluorescent light on the imaging screen are completely dark and cannot be seen, so that data as bright spots cannot be taken out. A spot having a large amount of fluorescent light is in a white state and the output overflows, so that a correct amount of fluorescent light cannot be measured. That is, it is possible to cope only with a change in the amount of fluorescence in a narrow range according to the dynamic range.
[0023]
In order to solve such a problem, it is conceivable that the CCD camera changes the gain and the exposure time to shoot an image, and then processes the image data to measure the fluorescence amounts of all spots. However, it is difficult for the CCD camera to correctly measure the amount of fluorescence between spots (probes 103) because dark noise is applied and S / N changes depending on the exposure time and gain.
[0024]
On the other hand, Patent Literature 2 discloses a method in which a light source irradiates a stable illumination light to a microarray chip without fluctuation and measures the fluorescence intensity of each spot by a photomultiplier. It only discloses controlling the amount of illumination light to be constant, but does not disclose anything about the case where the intensity of the fluorescence emitted from each spot changes.
[0025]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a light measuring device and a method capable of performing highly reliable and accurate light measurement in an always optimum state.
[0026]
[Means for Solving the Problems]
The invention according to claim 1 is arranged on a light source, a sample irradiated with light from the light source, light detection means for detecting the intensity of light from the sample, and an optical path between the light source and the sample. A light amount adjusting unit that makes it possible to adjust an amount of light irradiated on the sample from the light source, and the light amount adjusting unit so that the intensity of light detected by the light detecting unit becomes a predetermined specified value. Control means for controlling the amount of light applied to the sample, and light amount adjustment information by the light amount adjustment means when the intensity of light detected by the light detection means under the control of the control means matches the specified value. Output correction means for correcting the detection result of the light detection means based on the detection result. According to a second aspect of the present invention, in the first aspect of the present invention, the light amount adjusting means comprises an optical element capable of continuously or stepwise adjusting the amount of light applied to the sample. I have.
[0027]
According to a third aspect of the present invention, in the first aspect of the present invention, the light amount adjusting means includes a plurality of minute deflecting elements which are arranged at optically conjugate positions with the sample and which can selectively control deflection. It is characterized by comprising an optical deflecting element array in which the amount of light emitted from the light source to the sample can be adjusted according to the deflection control of these minute deflecting elements.
[0028]
According to a fourth aspect of the present invention, in the third aspect of the present invention, the sample comprises a microarray chip for detecting a reaction between a sample in the reaction solution and a probe, and further includes a movement for moving the reaction solution in the test site. Means for adjusting the amount of light applied to the sample by the light deflection element array in synchronization with the movement of the reaction solution by the movement means.
[0029]
The invention according to claim 5 irradiates a sample with light from a light source, detects the intensity of light from the sample with a light detection unit, and adjusts the amount of light irradiated on the sample from the light source with a light amount adjustment unit. Adjustable and controls the amount of light applied to the sample by the light amount adjusting means so that the intensity of light detected by the light detecting means becomes a preset specified value. The detection result of the light detection unit is corrected based on light amount adjustment information by the light amount adjustment unit when the intensity of light detected by the light detection unit matches the specified value.
[0030]
As a result, according to the present invention, the light detecting means can always perform highly reliable fluorescence measurement in an optimal state over a wide range without being affected by the dynamic range.
[0031]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0032]
(First Embodiment)
FIG. 1 shows a schematic configuration of a light measuring device to which the present invention is applied. In the figure, reference numeral 1 denotes a light source for illumination, and a deflection beam splitter 5 is disposed on an optical path of illumination light emitted from the light source 1 via a collector lens 2, a relay lens 3 of an illumination optical system, and an excitation filter 4. ing. The collector lens 2 converts illumination light from the light source 1 into parallel light. The excitation filter 4 is for selecting excitation light for fluorescence observation from illumination light. The deflection beam splitter 5 reflects the excitation light from the excitation filter 4 and transmits the fluorescence from the microarray chip 7 described later.
[0033]
A microarray chip 7 as a sample is disposed on the reflection optical path of the deflection beam splitter 5 via an objective lens 6.
[0034]
The microarray chip 7 is mounted on a stage 8. The stage 8 can move the microarray chip 7 to a position designated by driving an X motor 10 and a Y motor 11 connected to a controller 9.
[0035]
For example, FIGS. 2A and 2B show a schematic configuration of a microarray chip 7 to which the present invention can be applied.
[0036]
In FIG. 2A, reference numeral 701 denotes a microarray chip main body. The microarray chip main body 701 has a rectangular parallelepiped shape, and has a protruding portion 701a formed along the front width direction. A plurality (four in the illustrated example) of test sites 702 are opened on the upper surface of the microarray chip main body 701. Further, a plurality of (four in the illustrated example) suction / discharge holes 703 corresponding to the test sites 702 are formed on the front surface of the protruding portion 701 a of the microarray chip main body 701. These suction / discharge holes 703 communicate with the openings of the corresponding test sites 702 inside the microarray chip main body 701.
[0037]
At the bottom of the test site 702 (a hole is formed at the bottom), a substrate 704 made of an aluminum anodic oxide film as shown in FIG. On the substrate 704, probes 705 such as cDNA and oligo DNA are spotted in a matrix at a high density (interval of several hundreds μm or less). To each test site 702, DNA (reaction solution) extracted from a specimen cell having a genetic disease labeled with a fluorescent dye is dropped by a pipette or the like, so that the DNA of the specimen and the probe are hybridized.
[0038]
In this case, in order to increase the hybridization speed between the probe and the reaction solution dropped on the test site 702, the pump 14 shown in FIG. It is connected. That is, the reaction solution is suctioned and discharged by the pump 14 so that the reaction solution is repeatedly passed through the substrate 704 to increase the hybridization speed.
[0039]
Returning to FIG. 1, a photomultiplier (PMT) 13 as a light detecting unit is disposed on a transmission optical path of the deflection beam splitter 5 via an imaging lens 12. The imaging lens 12 guides the fluorescence emitted from the microarray chip 7 to the photomultiplier 13. Upon receiving the fluorescent light, the photomultiplier 13 outputs a minute pulse-like current waveform as a signal O according to the intensity of the fluorescent light.
[0040]
A power supply unit 16 is connected to the photo multiplier 13. The power supply unit 16 can change the drive voltage (HV: high volt) of the photomultiplier 13. In this embodiment, a voltage having the highest quantum efficiency is supplied as a fixed value to the photomultiplier 13 as a drive voltage.
[0041]
In the optical path between the collector lens 2 and the relay lens 3, a rotary ND filter 17 as a light amount adjusting means is arranged. The ND filter 17 has a disk shape as shown in FIG. 3, and its plate surface is configured to continuously change the light transmittance (ND value) along the circumferential direction. . In the illustrated example, a filter having a different light transmittance (ND value) depending on the shading of a color is shown, and a light-colored portion indicates a filter portion having the largest light transmittance, and a dark-colored portion indicates a filter portion having the smallest light transmittance. ing. Therefore, by rotating such an ND filter 17, the light transmittance (ND value) can be varied stepwise or continuously.
[0042]
In this embodiment, a rotary ND filter 17 requiring a characteristic that continuously changes the ND value is used. Of course, the change of the ND value may be stepwise.
[0043]
A stepping motor 18 is connected to the ND filter 17 via a rotation shaft 181. As the stepping motor 18, a bipolar two-pole motor is used.
[0044]
The stepping motor 18 is provided with a rotary position detecting sensor plate 19. The position detecting sensor plate 19 is fixed to a rotating shaft 182 connected with the rotating shaft 181 of the stepping motor 18 so as to have the same axial center, and is rotated completely in synchronization with the rotation of the ND filter 17.
[0045]
The position detecting sensor plate 19 is provided with a slit 191 at only one position on the peripheral surface, and the position of the slit 191 is set as a reference position.
[0046]
A photo interrupter 20 is provided close to the position detection sensor plate 19. The photo interrupter 20 detects the slit 191 of the position detecting sensor plate 19, and outputs this detection signal as a signal R indicating that the ND filter 17 is at the origin position. Here, the case where one rotary ND filter is used has been described, but a plurality of filters may be used in combination, or a slide ND filter may be used.
[0047]
The photomultiplier 13, the stepping motor 18, and the photointerrupter 20 are connected to a control unit 21 as control means. The control unit 21 analyzes the signal O output from the photomultiplier 13 and determines whether or not the signal O matches the specified value. Based on these matches, an ND address-fluorescence amount conversion process (output correction unit) is performed as an output correction process. After that, the signal M for driving the stepping motor 18 is output, and the signal R from the photo interrupter 20 is detected to detect the origin position of the ND filter 17.
[0048]
A computer (PC) 22 is connected to the control unit 21. The computer 22 has a function of outputting a control signal C to the control unit 21 and taking in the fluorescence intensity signal F output from the control unit 21 and displaying the signal on a monitor screen (not shown). An instruction signal S for moving the stage 8 to the designated position is output to the controller 9, and a control signal P for instructing the pump 14 to perform a suction / discharge operation is output.
[0049]
FIG. 4 shows a schematic configuration of the control unit 21. In the figure, reference numeral 211 denotes a current-to-voltage conversion circuit having a buffer function. The current-to-voltage conversion circuit 211 includes an operational amplifier, a capacitor, and the like (not shown), and outputs a pulse-shaped current waveform signal output from the photo-interrupter 20. The waveform band of O is dulled and converted to a voltage output V. An A / D converter 212 is connected to the current-voltage conversion circuit 211. The A / D converter 212 samples the input voltage output V at the timing of the sampling clock CLK and outputs it as a 10-bit digital signal (D0 to D9). The control unit 213 is connected to the A / D converter 212. The control unit 213 controls the entire operation of the control unit 21 and is designed with an FPGA.
[0050]
The control unit 213 incorporates a magnitude comparator (not shown) for comparing reference output value data set in advance as a reference value with the output of the photomultiplier 13 output from the A / D converter 212. ing.
[0051]
Further, the control unit 213 outputs six drive signals (M1 to M6) for driving the stepping motor 18. These drive signals (M1 to M6) are sent to the motor driver 215. The motor driver 215 includes a MOS-FET (not shown) or the like, and amplifies the drive signals (M1 to M6) output from the control unit 213 and outputs the amplified drive signals to the stepping motor 18 as OM1 to OM6. .
[0052]
Further, the control unit 213 outputs the fluorescence intensity signal F to the computer 22 as 12-bit data, receives an instruction signal S corresponding to various commands from the computer 22, and appropriately executes a necessary sequence. Has become.
[0053]
Further, upon receiving a signal R indicating that the ND filter 17 is at the origin position from the photo interrupter 20, the control unit 213 resets an internal address counter (not shown). This address counter is a 16-bit address counter, and enables both up-counting and down-counting.
[0054]
The clock source 214 is connected to the control unit 213. The clock source 214 drives the FPGA of the control unit 213, and outputs a clock signal of, for example, 40 MHz.
[0055]
Next, the operation of the first embodiment configured as described above will be described.
[0056]
Here, the description of the principle of the hybridizing operation, which is not directly related to the present embodiment, is omitted.
[0057]
In this case, when the hybridization operation ends, the flowchart shown in FIG. 5 is executed.
[0058]
First, the control unit 21 performs initialization in step 501. in this case. An origin detection operation for bringing the ND filter 17 to the reference position is started. The control unit 21 detects the signal level of the photo interrupter 20 and determines whether the current state is the origin position. When the slit 191 of the position detecting sensor plate 19 is at the position of the photo interrupter 20, the photo interrupter 20 outputs the L level.
[0059]
If the photo interrupter 20 and the slit 191 do not coincide with each other as shown in the example in the figure, the photo interrupter 20 becomes H level and it is known that the photo interrupter 20 has not come to the origin position. The signal M is output to rotate the stepping motor 18. Thus, the ND filter 17 is also rotated in synchronization with the rotation of the stepping motor 18. Then, when the ND filter 17 reaches the origin position and the signal R from the photo interrupter 20 becomes L level, the control unit 21 stops the operation of the stepping motor 18. Next, the control unit 21 resets an address counter (not shown) inside the control unit 213.
[0060]
In the present embodiment, the origin position of the ND filter 17 is located at an intermediate position in the variable range of the ND value.
[0061]
Next, the computer 22 outputs an instruction signal S to the controller 9 to move the stage 8 to the origin position (Step 502). Subsequently, when the user instructs the start of measurement from a keyboard (not shown) of the computer 22, one of the test sites 702 of the microarray chip 7 on the stage 8 is designated, and at the same time, the test sites 702 are arranged in a matrix in the test site 702. One of the spots (probe 705) is designated, and the stage 8 is moved to a position where the fluorescence luminance of this spot (probe 705) can be measured.
[0062]
If it is confirmed in step 503 that the stage 8 has moved to the designated position, the light source 1 emits illumination light. The illumination light enters the collector lens 2 and becomes parallel light, passes through the ND filter 17 and the relay lens 3, and enters the excitation filter 4. Then, excitation light is selected by the excitation filter 4, reflected by the deflection beam splitter 5, and condensed by the objective lens 6 on a specified probe 705 inside a specified test site 702 of the microarray chip 7.
[0063]
Then, in the spot (probe 705), when the fluorescent dye of the labeled sample is excited and emits fluorescence, the fluorescence passes through the objective lens 6 and the deflection beam splitter 5, and the photomultiplier is formed by the imaging lens 12. 13 is incident.
[0064]
The computer 22 outputs a sampling start command for sampling the pulsed current waveform signal O output from the photomultiplier 13 to the control unit 21 as a control signal C (step 504).
[0065]
Upon receiving the sampling start instruction, the control unit 21 outputs CLK to the A / D converter 212 from the control unit 213 and starts sampling. When sampling is started, the A / D converter 212 outputs the current output value from the photomultiplier 13 to the control unit 213 as 10-bit digital data.
[0066]
The control unit 213 compares the 10-bit digital data from the A / D converter 212 with reference output value data set in the control unit 213 as a specified value in advance (step 505).
[0067]
Here, when the data of the A / D converter 212 is larger than the reference output value data, that is, when the output of the photomultiplier 13 is large (step 506), the control unit 213 reduces the light transmittance of the ND filter 17. For this purpose, a drive signal (M1 to M6) for rotating the stepping motor 18 clockwise is output (step 507).
[0068]
The drive signals (M1 to M6) are current-amplified by the motor driver 215, become signals OM1 to OM6, and rotate the stepping motor 18. At the same time, the control unit 213 counts down the internal address counter by the amount of the operation of the stepping motor 18 (step 508). With such an operation, the amount of excitation light condensed on the spot (probe 705) from the light source 1 through the ND filter 17 decreases, the luminance of fluorescence decreases, and the output of the photomultiplier 13 decreases. .
[0069]
On the other hand, when the data of the A / D converter 212 is smaller than the reference output value data, that is, when the output of the photomultiplier 13 is small (step 509), the control unit 213 increases the light transmittance of the ND filter 17. Then, drive signals (M1 to M6) for rotating the stepping motor 18 counterclockwise are output (step 510).
[0070]
The drive signals (M1 to M6) are current-amplified by the motor driver 215, become signals OM1 to OM6, and rotate the stepping motor 18. At the same time, the control section 213 counts up the internal address counter by the amount that the stepping motor 18 is operated (step 511). By such an operation, the amount of excitation light condensed on the spot (probe 705) from the light source 1 through the ND filter 17 increases, the luminance of the fluorescence increases, and the output of the photomultiplier 13 increases. .
[0071]
Accordingly, if the output of the photomultiplier 13 shown in FIG. 6A is too large than the reference output value data (specified value), the ND filter 17 shown in FIG. While reducing the rate, the address counter shown in FIG. 4C is counted down (period T1). During this operation, the amount of excitation light condensed on the spot (probe 705) decreases, the luminance of the fluorescence decreases, and the output of the photomultiplier 13 decreases. However, as a result, when the output of the photomultiplier 13 falls too much below the reference output value data (specified value), the ND filter 17 shown in FIG. At the same time, the address counter shown in FIG. 3C is counted up (period T2). During this operation, the amount of the excitation light focused on the probe 705 increases, the luminance of the fluorescent light increases, and the output of the photomultiplier 13 also increases. Hereinafter, such an operation is repeatedly performed after the period T3, and the process ends after waiting for a state where the output of the photomultiplier 13 matches the reference output value data (specified value) shown in the period T4.
[0072]
If the output of the photomultiplier 13 matches the reference output value data (specified value) in step 512, ND address-fluorescence amount conversion processing is performed as output correction processing (step 513). Output as data F. Here, the output of the photomultiplier 13 when the ND filter 17 is at the origin position, that is, the value of the address counter when the digital data of the A / D converter 212 matches the reference output value data (specified value) becomes 0. Then, the fluorescent luminance at this time is output as data F, which is 2048 in decimal (1000 0000 0000 0000 in 12 bits).
[0073]
Thereby, when the output of the photomultiplier 13 matches the reference output value data (specified value), if the value of the address counter is smaller than 0, the fluorescence luminance data is larger than 2048, that is, the fluorescence is Data F is output to lower the light transmittance of the ND filter 17 because it is bright. For example, in the present embodiment, when the value of the address counter is -16384, the fluorescent brightness data is 3072 because it is 1.5 times brighter than the above-described fluorescent brightness data. When the value of the address counter is larger than 0, the result is the reverse of the above. For example, when the value of the address counter is 16384, the brightness becomes half of the above-described fluorescence luminance data. The fluorescent luminance is 1024.
[0074]
These fluorescent luminances are output to the computer 22 as fluorescent light amount data F (step 514).
[0075]
Thereafter, for the other spots (probes 705) in the same test site 702 for which fluorescence measurement is to be performed, the above-described measurement operation is repeated while moving the stage 8 to a position where each spot (probe 705) can be measured. The measurement is repeated for the test site 702 in the same manner. That is, the above-described measurement operation is sequentially performed on the spot (probe 705) for which fluorescence is to be measured in the same test site 702, and then the process moves to the next test site 702, and the spot (probe 705) for which the fluorescence luminance is to be measured is similarly set. On the other hand, the measurement operation described above is repeated, and the measurement operation is performed for all test sites 702.
[0076]
Then, when it is determined in step 515 that the measurement of the fluorescence luminance for all the test sites 702 and spots (probes 705) has been completed, the processing is terminated.
[0077]
Therefore, according to this configuration, the light transmittance of the ND filter 17 arranged in the optical path is obtained from the output of the photomultiplier 13 according to the fluorescent luminance from the spot (probe 705), a preset reference value, and the comparison result. And the output of the photomultiplier 13 is constantly controlled to be constant, and the output correction of the photomultiplier 13 such as a fluorescence amount conversion process is performed based on the data of the light transmittance of the ND filter 17 at this time. I have. Accordingly, the photomultiplier 13 can always perform highly reliable and accurate fluorescence measurement in an optimal state over a wide range without being affected by the dynamic range. In particular, with respect to the photomultiplier 13, the driving voltage (HV: high volt) with the highest quantum efficiency can be set as a fixed value, so that a more stable and highly accurate fluorescence measurement can be realized.
[0078]
Further, such a concept can be applied not only to the influence of the dynamic range of the detector such as the photomultiplier 13 but also to the case where the luminous efficiency of the fluorescent dye is nonlinear.
[0079]
In the above-described embodiment, the ND filter 17 is of a rotary type, but there is no problem in using a mechanism for inserting a plurality of ND filters having ND values in the optical path. In this case, a function for managing the ND value of the ND filter to be inserted and the number of inserted ND filters may be provided.
[0080]
Further, in the above-described embodiment, an operation of acquiring data after hybridization has been mainly described. However, data in the middle of hybridization may be acquired. In this case, the computer 22 operates the control unit 21, the pump 14, and the like so as to execute the operation of analyzing the fluorescence luminance of a desired spot (probe 705) once for each operation of the pump 14. Is possible without any difficulty.
[0081]
Further, in the above-described embodiment, the microarray chip 7 using an aluminum anodic oxide film as the substrate has been described. However, the substrate of the microarray chip is not particularly limited, and may be a two-dimensional substrate such as a silicon wafer or glass. In addition, various porous substrates can be used. In particular, when a porous substrate is used as the substrate of the microarray chip, the detection can be performed while moving the reaction solution in the test site 702 by using the pump 14 to perform the hybridization reaction, which is more preferable. .
[0082]
Furthermore, in the above-described embodiment, the ND filter 17 is used, and the light transmittance of the ND filter 17 is adjusted according to the output of the photomultiplier 13. However, for example, as shown in FIG. The aperture stop 23 is arranged at the pupil position of the lens 6, and the control unit 21 controls the opening degree of the aperture stop 23 so that the output of the photomultiplier 13 matches the reference output value data (specified value). The fluorescence amount conversion processing may be performed on the output of the photomultiplier 13 based on the data of the opening degree of the aperture stop. Alternatively, it is also possible to adopt a method in which the lighting method of the illumination 1 is changed to a pulse lighting method and the lighting duty is changed to adjust the amount of illumination applied to the microarray chip. The lighting duty is controlled so that the output of the photomultiplier 13 matches the reference output value data (reference value). In this state, the output of the photomultiplier 13 is subjected to a fluorescence amount conversion process based on the lighting duty data. It may be.
[0083]
(Second embodiment)
Next, a second embodiment of the present invention will be described.
[0084]
FIG. 7 shows a schematic configuration of the second embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0085]
In this case, a reflection mirror 31 is disposed on the optical path of the illumination light emitted from the light source 1 via the collector lens 2. An optical deflecting element array as a light amount adjusting unit, for example, a DMD (Digital Micromirror Device) 32 is arranged on the reflection optical path of the reflection mirror 31. Details of the DMD 32 will be described later.
[0086]
A dichroic mirror 33 is disposed on the reflection optical path of the DMD 32 (direction B in the drawing) via a relay lens 3 and an excitation filter 4. The dichroic mirror 33 reflects the excitation light from the excitation filter 4 and transmits the fluorescence from the microarray chip 7.
[0087]
A microarray chip 7 (see FIG. 2 for details) is disposed on the reflected light path of the dichroic mirror 33 via an objective lens 6. In addition, a CCD camera 34 as a light detecting unit is disposed on the transmitted light path of the dichroic mirror 33 via the imaging lens 12.
[0088]
The CCD camera 34 is connected to a dedicated PCI board (not shown) mounted on the computer 22 via a cable, and receives instruction data such as an imaging start timing and a shutter speed from the computer 22. It is designed to be sent directly.
[0089]
In this embodiment, the gain, shutter speed, and the like of the CCD camera 34 are set as fixed values based on characteristics such as the S / N ratio of the camera itself. The image data output to the computer 22 is sent as 12-bit data. The power of the CCD camera 34 is supplied from a PCI board (not shown).
[0090]
FIG. 8 shows a schematic configuration of the DMD 32.
[0091]
The DMD 32 is, for example, as disclosed in Patent Document 3, and has a landing chip 322, a yoke 323, and a hinge 324 on a CMOS substrate 321 as shown in FIG. Micro mirror 325 is attached. The CMOS substrate 321 has the same principle and structure as an SRAM generally known as a memory. The mirror 325 is turned on / off by the same operation as writing 1 or 0 to a specified memory address. You can make it. Normally, when the mirror 325 is off, the two mirrors 325 shown in FIG. 8 are on the left side (−10 degrees). When the mirror 325 is on, the two mirrors 325 shown in FIG. The mirror 325 is on the right side (+10 degrees). The mirrors 325 capable of such control are configured in a matrix of, for example, 1000 × 1000, so that on / off control can be performed on each mirror 325 independently. .
[0092]
Such a DMD 32 is disposed at an optically conjugate position of the microarray chip 7 so that the plurality of micro mirrors 325 are projected perpendicular to the illumination optical axis and perpendicular to the optical axis of the objective lens 6. I have. The mirrors 325 are selectively turned on / off independently at a response speed of the order of 10 μsec by a drive signal Q from the control unit 35. In the drawing, when the mirror 325 is in the on state, the light emitted from the light source 1 is directed in the B direction to enable illumination of the microarray chip 7, and in the off state, the light is directed in the A direction. The microarray chip 7 cannot be illuminated.
[0093]
9A and 9B show an actual operation state of the mirror 325, FIG. 9A shows an off state, and FIG. 9B shows an on state.
[0094]
FIG. 10 shows the on / off state of the mirror 325 and the direction of light reflected by the mirror 325. That is, FIG. 11A shows an off state, in which incident light is reflected in the direction A, and the microarray chip 7 cannot be illuminated. FIG. 10B shows an on state, and the incident light is B The light is reflected in the direction, and the microarray chip 7 can be illuminated.
[0095]
FIG. 11 shows the light amount adjustment by the on / off operation of the DMD 32. In this case, the mirror operation of the DMD 32 performs the PWM control for adjusting the light amount by changing the on / off duty every cycle (1 / f). Here, as shown in FIG. 7A, when the ON time of the mirror 325 is long, the light emitted from the light source 1 can illuminate the microarray chip 7 for a long time, and as shown in FIG. When the on time of the mirror 325 is short, the light emitted from the light source 1 illuminates the microarray chip 7 for a short time.
[0096]
In summary, when the on-time of the mirror 325 is long as shown in FIG. 12A, the light irradiated on the microarray chip 7 is strong, and conversely, as shown in FIG. When the on-time is short, the light applied to the microarray chip 7 becomes weak. By performing the PWM control of the mirror 325 in this manner, the energy of the illumination applied to the microarray chip 7 per unit time, that is, the light amount Can be adjusted.
[0097]
Note that details of the driving method of the DMD 32 and the like are disclosed in Patent Literature 4 and the like, and are omitted here.
[0098]
Returning to FIG. 7, the stage 8 on which the microarray chip 7 is mounted can be moved in a one-dimensional direction by an X motor 10 connected to a controller 9. The controller 9 drives the X motor 10 based on an instruction from the computer 22.
[0099]
A pump 14 is connected to the microarray chip 7 via a Teflon tube 15. The pump 14 is connected to the computer 22 via the control line P, and in response to an instruction from the computer 22, sucks and discharges the reaction solution to and from the microarray chip 7, and repeatedly passes the reaction solution to the substrate 704. In this way, hybridization is performed.
[0100]
Next, the operation of the first embodiment configured as described above will be described.
[0101]
In this case, when the hybridization operation ends, the flowchart shown in FIG. 13 is executed.
[0102]
First, the user designates a desired test site 702 and a spot (probe 705) of the microarray chip 7 from a keyboard (not shown) connected to the computer 22, and subsequently instructs a start of fluorescence measurement. Here, a batch measurement of all spots (probes 705) on the test site 702 is instructed.
[0103]
First, the computer 22 outputs the instruction signal S to the controller 9, drives the X motor 10, and moves the stage 8 to a position where the fluorescence of the spot (probe 705) of the specified test site 702 of the microarray chip 7 can be measured. (Steps 1301 and 1302).
[0104]
Next, the computer 22 outputs a spot address to the control unit 35 via the signal line C in order to irradiate only the spot (probe 705) at which fluorescence luminance measurement is started with illumination light. The control unit P also outputs an instruction for the suction / discharge operation of the pump 14 to the pump 14.
[0105]
The pump 14 sucks or pushes out the reaction solution in the test site 702 in accordance with an operation instruction through the control line P, and moves the solution in and out of the alumina substrate 704. That is, the reaction solution in the test site 702 repeats the filled state shown in FIG. 14A and the removed state shown in FIG. 14B.
[0106]
In this state, the computer 22 instructs the pump 14 to stop the cylinder (not shown) in the suction state. By doing so, the reaction solution dropped on the test site 702 of the microarray chip 7 is drawn toward the pump 14, and the reacted spot (probe 705) is exposed (see FIG. 14B).
[0107]
Since this state is most suitable for fluorescence luminance measurement, the computer 22 performs PWM control on the mirror 325 of the DMD 32 to irradiate the spot (probe 705) with light. On the other hand, in the state shown in FIG. 14A in which the reaction solution is filled in the test site 702, the computer 22 turns off the mirror 325 of the DMD 32 so as not to shine light on the spot (probe 705). I try to make it.
[0108]
With the computer 22 controlling the mirror 325 of the DMD 32 by the PWM control, the fluorescence luminance measurement is started.
[0109]
In this case, the illumination light emitted from the light source 1 enters the collector lens 2 and becomes parallel light, is reflected by the reflection mirror 31, and enters the DMD 32. Then, the reflected light determined by the PWM control of the DMD 32 passes through the relay lens 3 and enters the excitation filter 4. Then, the excitation light is selected by the excitation filter 4, reflected by the dichroic mirror 33, and focused on the spot (probe 705) of the specified test site 702 of the microarray chip 7 by the objective lens 6.
[0110]
Then, when the fluorescent dye of the labeled sample is excited at the spot (probe 705) and emits fluorescence, the fluorescence passes through the objective lens 6 and the dichroic mirror 33, and is transmitted to the CCD camera 34 by the imaging lens 12. An image is formed on an imaging surface (not shown).
[0111]
In this case, the PWM control of the DMD 32 is instructed by the drive signal Q from the control unit 35.
[0112]
FIG. 15 shows the configuration of the control unit 35. In this case, the signal line C is composed of 10 bits, the upper 2 bits instruct the ON / OFF operation of the mirror 325 of the DMD 32 and the change of the duty, and the lower 8 bits instruct the position information of the spot (probe 705). Things. The lower 8 bit signal line is connected to the mirror address generator 351. For example, when it is desired to illuminate the position of the first spot (probe 705), the mirror address generator 351 calculates the addresses of the plurality of mirrors 325 of the DMD 32 that illuminates only the spot (probe 705), and the frame memory 352a of the DMD driver 352. Is transmitted in the form of image data.
[0113]
In this case, the relationship between the spot (probe 705) whose fluorescence luminance is measured and the plurality of mirrors 325 of the DMD 32 is as shown in FIG. Here, only a part of the entire DMD 32, that is, the mirror 325 in the area shown in FIG. That is, the spot (probe 705) is irradiated with light in the form of a spot by the plurality of mirrors 325 in the area 32a designated to be turned on.
[0114]
Similarly, the mirror 325 in the entire area of the DMD 32 is divided into a plurality of areas, these areas are allocated to a large number of spots (probes 705), and the corresponding spots (probes) are designated by turning on the mirror 325 in each area. 705) is irradiated with spot-shaped light.
[0115]
The DMD 32 is composed of a 1000 × 1000 micromirror group, and the frame memory 352 a inside the DMD driver 352 stores on / off data of all the mirrors 325 of 1000 × 1000 like a video memory. The DMD driver 352 is driven by the clock 353, generates a drive signal Q for driving the DMD 32 based on the contents of the frame memory 352a, and transmits the drive signal Q to the DMD 32. In addition, the DMD driver 352 outputs duty information for turning on / off the mirror group that irradiates the spot (probe 705) with light, that is, the mirror 325 in the area 32a shown in FIG. In this case, in the present embodiment, 12 bits are used in order to enable PWM control in 4096 steps. This duty information is output to the computer 22 as a signal F.
[0116]
In this state, when an imaging instruction command is transmitted from the computer 22 to the CCD camera 34 via the signal line V, the imaging by the CCD camera 34 is started (step 1303). At the same time, the computer 22 sends the position information of the spot (probe 705) for which the fluorescence luminance is to be measured to the control unit 35 via the signal line C to the control unit 35 at the duty of 50% as a default in the area 32a shown in FIG. The mirror 325 is instructed to be turned on / off (step 1304).
[0117]
In this state, when the CCD camera 34 starts imaging and image data is sent to the computer 22 via the signal line V, the CPU (not shown) of the computer 22 stores one image in a video memory (not shown) inside. The image data for each minute is stored.
[0118]
When the storage in the video memory is completed, the CPU determines the luminance information of the address corresponding to the spot (probe 705) for measuring the fluorescent luminance in the video memory based on the positional information of each spot (probe 705) which is stored in advance as data. Read.
[0119]
Then, the brightness information acquired by the CCD camera 34 is compared with a preset specified value (step 1305). Here, when the fluorescence luminance acquired by the CCD camera 34 is smaller than a predetermined value (step 1306), the computer 22 instructs a duty change via the signal line C to increase the on-time of the mirror 325 group. It is sent (step 1307). On the other hand, if the fluorescent luminance acquired by the CCD camera 34 is larger than the predetermined value (step 1308), the computer 22 sends an instruction to change the duty via the signal line C so as to reduce the on-time of the group of mirrors 325. (Step 1309).
[0120]
When the fluorescent brightness of the spot (probe 705) obtained by the CCD camera 34 reaches a specified value (step 1310), the computer 22 converts the duty to fluorescent brightness information via the signal F and repeats such an operation. It is displayed on a monitor (not shown) (step 1311).
[0121]
In this case, for example, if the signal F is numerical data of 3864 representing a duty of 90%, the amount of fluorescence of the spot itself is very small, and 410 which is 10% of 4096 is displayed on the computer 22 monitor. . If the signal F is numerical data of 410 representing a duty of 10%, the amount of fluorescence of the spot itself is very large, and 3864 which is 90% of 4096 is displayed on the monitor of the computer 22.
[0122]
Thereafter, for other spots (probes 705) in the same test site 702 where fluorescence measurement is to be performed, the mirrors 325 in other corresponding areas (addresses) of the DMD 32 are turned on, and the duty for turning on / off is set. Is set, the above-described operation of the fluorescence luminance measurement is repeatedly executed. Then, when the measurement of the fluorescent luminance of all the spots (probes 705) is completed (step 1312), the fluorescent luminance measurement is repeated for the other test sites 702 in the same manner. In this case, when measuring the fluorescent luminance for another test site 702, the X motor 10 is driven to move the stage 8 to a position where the fluorescent luminance can be measured for the next test site 702.
[0123]
Then, when it is determined that the fluorescence luminance measurement has been completed for all the test sites 702, the process is terminated (step 1313).
[0124]
Therefore, according to this, the brightness information acquired by the CCD camera 34 in response to the fluorescence from the spot (probe 705), the reference value set in advance, and the mirror 325 of the DMD 32 arranged in the optical path are compared with the comparison result. The on / off duty is changed to constantly control the luminance information of the CCD camera 34, and the luminance information of the CCD camera 34 is corrected based on the duty information of the DMD 32 at this time. Thus, the CCD camera 34 can perform highly reliable and accurate fluorescence measurement in an optimum state over a wide range without being affected by the dynamic range. Further, in particular, since the CCD camera 34 can perform photographing with the gain and shutter speed fixed in the area where the S / N is the best, it is possible to realize highly accurate fluorescence luminance measurement in a more stable state. Further, since the CCD camera 34 can take an image corresponding to all the spots (probes 705) in units of the test sites 702 of the microarray chip 7, the stage 8 is moved to measure the fluorescence of each spot (probe 705). Such an operation can be omitted, the stage operation can be simplified, the control can be simplified accordingly, and the overall configuration can be simplified.
[0125]
Although the CCD camera 34 is used in the above-described embodiment, the photomultiplier (PMT) used in the first embodiment can be used without being limited to the CCD camera.
[0126]
Further, in the above-described embodiment, an operation of acquiring data after hybridization has been mainly described. However, data in the middle of hybridization may be acquired. In this case, the computer 22 may operate the control unit 21, the pump 14, and the like so as to execute an operation of analyzing the fluorescence luminance of a desired spot (probe 705) once for one operation of the pump 14. It is possible.
[0127]
Further, in the above-described embodiment, the mirror 325 of the DMD 32 is disposed at a position conjugate with the microarray chip 7 so that the mirror 325 in an area (address) corresponding to the position of the spot (probe 705) is subjected to PWM control. Although the fluorescence intensity of a desired spot (probe 705) is measured, the DMD 32 can be used as an aperture stop by further setting the DMD 32 at a position conjugate with the pupil of the objective lens 6. In this way, the DMD 32 arranged at a position conjugate with the microarray chip 7 turns on the mirror 325 in the area corresponding to the spot (probe 705) whose luminance is to be measured, and the DMD placed at a position conjugate with the pupil of the objective lens 6 If a mirror is selected in a circular or polygonal shape like an aperture stop and turned on with the mirror, fluorescence intensity measurement can be performed in the same manner as described above. This eliminates the need to perform the PWM control of the mirror 325, so that the drive circuit thereof can be omitted, contributing to a reduction in power consumption.
[0128]
Furthermore, in the above-described embodiment, the mirrors 325 of the respective regions assigned to the DMD 32 are sequentially turned on in correspondence with the spots (probes 705) to irradiate each spot (probe 705) with light. The mirrors 325 in each area may be turned on at the same time to irradiate light to each spot (probe 705) at the same time, and this state may be imaged by the CCD camera 34. In this case, the fluorescence measurement time can be significantly reduced.
[0129]
Furthermore, in the above-described embodiment, a microarray that performs a hybridization reaction using a nucleic acid as a probe has been described. However, a microarray that detects fluorescence by immobilizing an array of biologically related substances other than nucleic acid as a probe is described. Is also applicable. For example, examples of the probe include various proteins, antigens, antibodies, and the like.
[0130]
Furthermore, although the DMD is used in the above-described embodiment, a liquid crystal filter may be used instead.
[0131]
In addition, the present invention is not limited to the above-described embodiment, and can be variously modified in an implementation stage without departing from the spirit of the invention.
[0132]
Further, the embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent features. For example, even if some components are deleted from all the components shown in the embodiments, the problem described in the column of the problem to be solved by the invention can be solved, and the problem described in the column of the effect of the invention can be solved. In the case where the effect described above is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0133]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a light measuring device and a method capable of performing highly reliable and accurate light measurement in an always optimum state.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of an optical measurement device to which a first embodiment of the present invention is applied.
FIG. 2 is a diagram showing a schematic configuration of a microarray chip used in the first embodiment.
FIG. 3 is a diagram illustrating an ND filter used in the first embodiment.
FIG. 4 is a diagram showing a schematic configuration of a control unit used in the first embodiment.
FIG. 5 is a flowchart for explaining the operation of the first embodiment.
FIG. 6 is a time chart for explaining the operation of the first embodiment.
FIG. 7 is a diagram showing a schematic configuration of an optical measurement device to which a second embodiment of the present invention is applied.
FIG. 8 is a diagram showing a schematic configuration of a DMD used in the second embodiment.
FIG. 9 is a diagram showing an on / off state of a DMD used in the second embodiment.
FIG. 10 is a view for explaining the relationship between the on / off state of a DMD used in the second embodiment and the direction of light reflected by a mirror.
FIG. 11 is a view for explaining light amount adjustment by an on / off operation of a DMD used in the second embodiment.
FIG. 12 is a view for explaining that the amount of light can be adjusted by the on-time length of the DMD used in the second embodiment.
FIG. 13 is a flowchart for explaining the operation of the second embodiment.
FIG. 14 is a diagram illustrating a state of a reaction solution in a test site in the operation of the second embodiment.
FIG. 15 is a diagram showing a schematic configuration of a control unit used in the second embodiment.
FIG. 16 is a diagram for explaining the relationship between DMD mirror groups that irradiate light to a spot (probe) in the operation of the second embodiment.
FIG. 17 is a diagram showing a schematic configuration of a conventional microarray chip reader.
[Explanation of symbols]
1: light source, 2: collector lens, 3: relay lens
4: Excitation filter, 5: Deflection beam splitter
6 Objective lens 7 Microarray chip
701: Microarray chip body, 701a: Projecting portion
702: test site, 703: suction and discharge holes, 704: alumina substrate
705: Probe, 8: Stage, 9: Controller
10 X motor, 11 Y motor, 12 imaging lens
13 Photomultiplier, 14 Pump
15: Teflon tube, 16: Power supply unit
17 ... ND filter, 18 ... Stepping motor
181, 182: rotation axis, 19: sensor plate for position detection
191 ... Slit, 20 ... Photo interrupter
21: control unit, 211: current-voltage conversion circuit, 212: A / D converter
213: control unit, 214: clock source
215: motor driver, 22: computer, 31: reflection mirror
32: DMD, 32a: area, 321: CMOS substrate
322: landing tip, 323: yoke, 324: hinge
325: mirror, 33: dichroic mirror
34 ... CCD camera, 35 ... Control unit, 351 ... Mirror address generator
352: DMD driver, 352a: Frame memory, 353: Clock

Claims (5)

A light source,
A sample irradiated with light from the light source,
Light detection means for detecting the intensity of light from the sample,
Light amount adjusting means disposed in an optical path between the light source and the sample and capable of adjusting the amount of light emitted to the sample from the light source,
Control means for controlling the amount of light applied to the sample by the light amount adjustment means so that the intensity of light detected by the light detection means becomes a preset specified value; and Output correction means for correcting the detection result of the light detection means based on light amount adjustment information by the light amount adjustment means when the intensity of light detected by the light detection means matches the specified value. Light measuring device. 2. The light measuring device according to claim 1, wherein said light amount adjusting means comprises an optical element capable of continuously or stepwise adjusting the amount of light applied to said sample. The light amount adjusting means is disposed at an optically conjugate position with the sample, and has a plurality of minute deflecting elements that can be selectively controlled in deflection. The light source adjusts the light from the light source to the sample in accordance with the deflection control of these minute deflecting elements. 2. The optical measuring device according to claim 1, wherein the optical measuring device comprises an optical deflecting element array capable of adjusting an amount of irradiated light. The sample is composed of a microarray chip that detects a reaction between a sample and a probe in a reaction solution,
Further, a moving means for moving the reaction solution in the test site,
4. The optical measurement device according to claim 3, wherein the operation of adjusting the amount of light applied to the sample by the light deflection element array is synchronized with the movement of the reaction solution by the movement unit. Irradiate the sample with light from the light source,
The intensity of light from the sample is detected by light detection means,
The amount of light emitted from the light source to the sample can be adjusted by a light amount adjusting unit, and the light amount adjusting unit adjusts the intensity of the light detected by the light detecting unit to a predetermined value. Controlling the amount of light applied to the sample,
The control unit corrects a detection result of the light detection unit based on light amount adjustment information by the light amount adjustment unit when the intensity of light detected by the light detection unit matches the specified value. Measuring method.

Images