JP2010281842A - Device and method for measuring luminous energy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device and a method for measuring luminous energy which can quantitatively evaluate luminous energy. <P>SOLUTION: The luminous energy measuring device which measures luminous energy loaded with a light detection means is characterized by including a luminous energy calculating means calculating luminous energy of fluorescence signal light based on output signal of the light detection means, a light source for calibration which can control luminous energy of light given to the light detection means, and a calibrating means giving light of known luminous energy to the light detecting means with the light source for calibration to calibrate linearity of luminous energy calculated by the luminous energy calculating means. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a light amount measuring device and a light amount measuring method for measuring a light amount, and more particularly to a light amount measuring device and a light amount measuring method capable of quantitatively evaluating the light amount.

  A method using a microarray is known as a method for identifying a biopolymer such as DNA. For example, when DNA is identified, a DNA probe having a known base sequence is fixed to each site of the microarray, and DNA having a complementary base sequence is bound to each site by hybridization. By labeling the bound DNA with a fluorescent marker, the amount of binding can be recognized as the amount of fluorescence.

Toru Makino and Junichi Kano, Journal “Optics” Optical Technology in Life Science “DNA Analysis and Optical Technology”, Vol. 28, No. 10 (1999), Japan Society for Applied Physics, Japan Optical Society, 1999, p549-552

  The amount of light is measured using a dedicated light amount measuring device. However, since the absolute light quantity cannot be measured with the conventional apparatus, the gene expression level cannot be directly quantitatively evaluated.

  An object of the present invention is to provide a light quantity measuring device and a light quantity measuring method capable of quantitatively evaluating the quantity of light.

The light quantity measuring device of the present invention is a light quantity measuring device for measuring the light quantity of light taken in by the light detecting means, a light quantity calculating means for calculating a light quantity of fluorescent signal light based on an output signal of the light detecting means, and the light A calibration light source capable of controlling the amount of light applied to the detection means and a known light amount of light from the calibration light source to the light detection means to calibrate the linearity of the light quantity calculated by the light quantity calculation means. And a calibration means.
According to this light quantity measuring apparatus, the light quantity linearity calculated by the light quantity calculating means is calibrated by applying a known quantity of light to the light detecting means from the calibration light source, so that the light quantity can be quantitatively evaluated.

  The calibration light source may include a light source that outputs a fixed amount of light and a priced optical attenuator.

  You may provide the excitation light irradiation means to irradiate excitation light toward the target object which generate | occur | produces the said fluorescence signal light.

The light quantity measuring method of the present invention is a light quantity measuring method for measuring a light quantity of light taken in by a light detecting means, a step of calculating a light quantity of fluorescent signal light based on an output signal of the light detecting means, and the light detecting means Calibrating the linearity of the light amount calculated by the step of calculating the light amount by supplying light of a known light amount to the light detection means by a calibration light source capable of controlling the light amount of light applied to the light source. It is characterized by.
According to this light quantity measurement method, the light quantity linearity calculated by the light quantity calculation means is calibrated by applying light of a known light quantity to the light detection means from the calibration light source, so that the light quantity can be quantitatively evaluated.

  The calibration light source may include a light source that outputs a fixed amount of light and a priced optical attenuator.

  According to the light quantity measuring device of the present invention, the light quantity linearity calculated by the light quantity calculating means is calibrated by applying a known light quantity of light to the light detecting means from the calibration light source, so that the light quantity can be quantitatively evaluated. .

  According to the light quantity measuring method of the present invention, the light quantity linearity calculated by the light quantity calculating means is calibrated by applying a known light quantity of light to the light detecting means from the calibration light source, so that the light quantity can be quantitatively evaluated. .

The figure which shows the structure of the optical system of the light quantity measuring device of this embodiment. The block diagram which shows the structure of the control system of the light quantity measuring device of this embodiment.

  Hereinafter, with reference to FIG. 1 and FIG. 2, an embodiment of a light amount measuring apparatus according to the present invention will be described.

  FIG. 1 is a diagram illustrating a configuration of an optical system of a light amount measuring apparatus according to the present embodiment. The present embodiment is a light quantity measuring device that irradiates a DNA microarray (DNA or RNA sample on the microarray (hereinafter referred to as biochip)) with excitation light and measures the quantity of fluorescent signal light generated in the DNA microarray.

  As shown in FIG. 1, the light quantity measuring device of the present embodiment includes, as an optical system for irradiating a DNA microarray (biochip) 30 with excitation light, a green laser light source 1 and a red laser light source 2 that generate excitation light, , A mirror 3 for bending the irradiation light from the green laser light source 1, and a dichroic mirror 5, a microlens array 6, a mirror 7 and a dichroic mirror disposed in the optical paths of the irradiation light from the green laser light source 1 and the red laser light source 2, respectively. 8 and a movable mirror 9.

  The wavelengths of the green laser light source 1 and the red laser light source 2 match the excitation light of the fluorescent dyes cy3 and cy5.

  Moreover, the light quantity measuring device of this embodiment receives the relay lens 11 arranged in the optical path of the signal light and the light that has passed through the relay lens 11 as an optical system for receiving the fluorescent signal light generated in the DNA microarray 30. And a high-sensitivity CCD camera 12.

  Furthermore, the light quantity measuring device of the present embodiment uses a calibration laser light source 21 that outputs a laser beam for calibration as an optical system for calibrating the light quantity, and a laser beam output from the calibration laser light source 21. An optical attenuator 22 for attenuation. The calibration laser light source and the optical attenuator 22 may be attached at the time of calibration instead of being built in the apparatus.

  A calibration shutter 23 that blocks incident light on the CCD camera 12 is provided on the front side of the CCD camera 12.

  As shown in FIG. 1, the DNA microarray 30 is placed on a table 41.

  FIG. 2 is a block diagram showing the configuration of the control system of the light quantity measuring apparatus of this embodiment.

  As shown in FIG. 2, the light amount measuring device of the present embodiment includes a light amount calculation unit 51 that calculates the amount of light captured by the CCD camera 12, a calibration shutter drive unit 53 that drives the calibration shutter 23, and a mirror. 9, a mirror driving unit 55 that calibrates the light amount calculating unit 51, a green laser light source 1, a red laser light source 2, a CCD camera 12, a light amount calculating unit 51, a calibration laser light source 21, and an optical attenuator. 22, a calibration shutter drive unit 53, a mirror drive unit 55, and a control unit 61 that controls the calibration unit 56.

  Next, an operation during measurement of the DNA microarray 30 will be described.

  At the time of measurement, the movable mirror 9 and the calibration shutter 23 are driven to positions indicated by broken lines that are retracted from the optical path (FIG. 1).

  Laser light output from the green laser light source 1 or the red laser light source 2 is applied to the DNA microarray 30 via the mirror 3, the dichroic mirror 5, the microlens array 6, the mirror 7, and the dichroic mirror 8.

  The fluorescent signal light from the DNA microarray 30 generated by the excitation by the laser light enters the CCD camera 12 via the dichroic mirror 8 and the relay lens 11. The output signal of the CCD camera 12 is sent to the light amount calculation unit 51, and the light amount calculation unit 51 calculates the light amount of the signal light.

  For low-level signal light that is buried in background noise, random noise may be relatively reduced by averaging processing by repeated addition to improve the S / N ratio.

  In general, gene expression levels are distributed over a wide range, and in order to quantitatively measure gene expression levels, it is required to calculate the light quantity to a low level with high accuracy. However, the low-level light quantity is easily buried in the background light (background noise), and the calculation accuracy becomes a problem particularly in the low-level light quantity.

  Next, the operation at the time of calibration will be described.

  In this embodiment, the no-signal state of the CCD camera 12 is created and zero point adjustment is performed. At this time, the calibration shutter 23 is driven to a position indicated by a solid line that blocks the optical path (FIG. 1). Thereby, a state where stray light does not enter the CCD camera 12 is ensured. The output signal of the CCD camera 12 at this time is sent to the light amount calculation unit 51, and the calibration unit 56 performs calibration with respect to the light amount calculation unit 51 using the output signal value as a reference (zero point).

  Further, in the present embodiment, the amount of light incident on the CCD camera 12 is controlled to calibrate the light amount calculation particularly in the low light amount region. At this time, the calibration shutter 23 is driven to a position indicated by a broken line retracted from the optical path. Further, the movable mirror 9 is driven to a position indicated by a solid line entering the optical path (FIG. 1).

  As shown in FIG. 1, the light output from the calibration laser light source 21 is attenuated by the optical attenuator 22 and enters the CCD camera 12 via the movable mirror 9 and the relay lens 11. Since the amount of laser light output from the calibration laser light source 21 and the amount of attenuation by the optical attenuator 22 are known, the amount of light incident on the CCD camera 12 is also known. The laser light output from the calibration laser light source 21 has a wavelength close to the wavelength (570 nm) of the fluorescence signal light of cy3, for example.

  By switching the attenuation amount of the optical attenuator 22, for example, correctly valued light such as 0 dBm, −10 dBm, −20 dBm, or the like is incident on the CCD camera 12. The output signal of the CCD camera 12 at this time is sent to the light amount calculation unit 51, and the calibration unit 56 performs calibration on the light amount calculation unit 51 so that each output signal value corresponds to each light amount correctly.

  As described above, in this embodiment, the zero point is calibrated using the calibration shutter 23 and the attenuation of the optical attenuator 22 is switched, so that linearity correction of a particularly low level of light quantity can be performed. Therefore, the amount of light can be measured quantitatively and accurately up to a low level close to the background light. In addition, the amount of background light can be correctly evaluated.

  In the above embodiment, the calibration laser light source 21 and the optical attenuator 22 are used to give a known amount of light to the CCD camera 12, but a light source capable of adjusting the amount of light may be used instead. For example, an LED may be used as the light source and the amount of light may be controlled by the drive current.

Further, a white light source may be used as the calibration light source instead of the laser light source.
Further, instead of a combination of a calibration light source and an optical attenuator, an object (reference fluorescence signal generating means) that generates a reference fluorescence signal is installed instead of the biochip 30, and a green laser light source and a red laser light source ( Excitation light may be emitted from the excitation light irradiation means) and used as a calibration light source. Also, linearity calibration can be performed by preparing a plurality of objects that generate different reference fluorescence signals.

  In the above embodiment, the light amount is calibrated by accurately controlling the light amount of light incident on the CCD camera 12. However, the light amount incident on the CCD camera 12 may be accurately measured. For example, by attaching a power meter that can accurately measure the amount of light at the position of the CCD camera 12, the amount of light actually incident on the CCD camera 12 can be accurately measured, and the output signal of the CCD camera 12 under the same conditions can be obtained in advance. Calibration can be performed so as to correlate the measured light quantity. As a calibration method using such a power meter, the method disclosed in Japanese Patent Application Laid-Open No. 2004-191232 can be applied to the present invention.

  In the above embodiment, the calibration unit used for calibration is provided in the apparatus. However, a similar calibration method may be executed by attaching a calibration unit for calibration during calibration. Here, the calibration unit is a calibration light source 21, an optical attenuator 22, and a calibration shutter 23.

  In the above embodiment, the case where a DNA microarray is a measurement target has been exemplified, but the present invention can be similarly applied to detection of various biological substances such as proteins, sugar chains, metabolomes and the like. Further, the present invention is not limited to substance detection by a microarray, but can also be applied to evaluation of fluorescent dust in a semiconductor process, a fluorescent screen of a plasma display panel, and the like.

  The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a light quantity measuring device and a light quantity measuring method for calculating the quantity of fluorescent signal light based on the output signal of the light detection means.

1 Green laser light source (excitation light irradiation means)
2 Red laser light source (excitation light irradiation means)
12 CCD camera (light detection means)
21 Calibration Laser Light Source (Calibration Light Source)
22 Optical attenuator (light source for calibration)
23 Calibration shutter 51 Light quantity calculation unit (light quantity calculation means)
56 Calibration section (calibration means)

Claims (5)

In a light amount measuring device that measures the amount of light captured by the light detection means,
A light amount calculating means for calculating the light amount of the fluorescent signal light based on the output signal of the light detecting means;
A calibration light source capable of controlling the amount of light applied to the light detection means;
A calibration unit that calibrates the linearity of the light amount calculated by the light amount calculation unit by giving light of a known light amount to the light detection unit by the calibration light source;
A light quantity measuring device comprising:   The light quantity measuring device according to claim 1, wherein the calibration light source includes a light source that outputs a constant amount of light and a priced optical attenuator.   The light quantity measuring device according to claim 1, further comprising excitation light irradiation means for irradiating excitation light toward an object that generates the fluorescent signal light. In the light quantity measurement method for measuring the light quantity of light captured by the light detection means,
Calculating the amount of fluorescent signal light based on the output signal of the light detection means;
Calibrating the linearity of the light amount calculated by the step of calculating the light amount by applying light of a known light amount to the light detection unit by a calibration light source capable of controlling the light amount of light applied to the light detection unit; ,
A light quantity measuring method comprising:   5. The light quantity measuring method according to claim 4, wherein the calibration light source includes a light source that outputs light having a constant light quantity and a priced optical attenuator.

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