JP2007212478A - Light quantity measuring device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a light quantity measuring device which can measure not only shape but also absolute light quantity, while being able to directly estimate the number of fluorescence molecules, etc. <P>SOLUTION: This device is equipped with light quantity detector having one- or two-dimensionally-arranged photo acceptance units. The light quantity detector utilizes outgoing light beam from reference luminous source in which its own light quantity is measured to be valued by the power meter acquired traceability to national standard of optical power, calibrating relationship between received light quantity of each photo acceptance unit and its output signal all at once. Thus the device will measure absolute light quantity of fluorescence generated from biochip, cell, fluorescence coated object, or fluorescence dust. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a light quantity measuring device using a light quantity detector having light receiving elements arranged one-dimensionally or two-dimensionally and having traceability as a national standard of optical power. As a light quantity detector, it is a camera with the light-receiving part which consists of a some light receiving element, for example.

Conventionally, DNA chips are used for identification and fractionation of biopolymers (for example, detection of DNA, detection of the presence or absence of genetic DNA).
A DNA chip is obtained by, for example, fixing thousands of tens of thousands of known DNA fragments to sites on a substrate. When an unknown DNA fragment is passed through such a DNA chip, the same kind of DNAs hybridize and bind to each other. After the unknown DNA that has not been bound is removed by washing, the remaining DNA is irradiated with excitation light (laser light). The unknown DNA is preliminarily labeled with a fluorescent material, and fluorescence is generated from the fluorescent material excited by irradiation with excitation light. As a result, light and dark appear for each site corresponding to whether or not hybridization has occurred.

This bright / dark observation is performed by a fluorescence scanning apparatus using a confocal laser optical system (see, for example, Non-Patent Document 1). As a fluorescence observation detector, a photomultiplier (Photo multiplier) capable of high sensitivity detection is used.
In some cases, a power meter is used as a detector. However, since the power meter can only perform zero-dimensional detection, a sample having a shape such as a biochip such as a DNA chip or a plasma display is accurate. Cannot be measured. This is because the amount of fluorescent light has a spatial distribution depending on the shape of the sample.

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

By the way, detectors such as cameras and fluorescent scanning devices have the following problems because they cannot measure absolute light quantity.
(1) The value of the output signal of the detector cannot be compared between devices.
(2) In biochip measurement, since the gene expression level is unknown, there is no choice but to make a comparative measurement by mixing a known gene or the like, which makes the apparatus expensive.

  An object of the present invention is to solve the above-described problems, and to provide a light amount measuring apparatus capable of measuring an absolute light amount together with a shape and directly estimating the number of fluorescent molecules.

In order to achieve such an object, the invention of claim 1 has light receiving elements arranged one-dimensionally or two-dimensionally, and is measured by a power meter that is traceable to the national standard of optical power. A light amount detector in which the relationship between the received light amount of each light receiving element and its output signal is calibrated at a time using the emitted light from the illumination reference light source for which the light amount is valued, is provided with a biochip or cell or The absolute light quantity of the fluorescence generated from the fluorescent coated body or the fluorescent dust is measured.
According to such a light quantity measuring device, it is possible to measure an absolute light quantity with a light quantity detector. Moreover, as a light quantity detector, the camera with the light-receiving part which consists of a some light receiving element can be used.

  Further, the invention of claim 2 is an illumination having light receiving elements arranged in a one-dimensional or two-dimensional manner, and the amount of light of which is measured by a power meter that is traceable to the national standard of optical power. A light amount detector in which the relationship between the received light amount of each light receiving element and its output signal is calibrated at a time using the emitted light from the reference light source, and the light amount detector that detects the fluorescent light amount from the fluorescent object. Means for calculating the number of molecules of the fluorescent object from an output signal and a relational expression of the amount of light generated from the fluorescent dye, and directly estimating the light quantity or the number of molecules or the quantity of light and the number of molecules of the fluorescent object. And

The present invention has the following effects.
(1) Since a camera having a light receiving unit in which light receiving elements are arranged is calibrated with respect to each light receiving element by a power meter traceable from national standards, the absolute light quantity can be directly measured from the camera output.
(2) If the camera calibrated in this way is used, it is possible to easily reduce the light amount measurement error between the respective apparatuses for fluorescence measurement.

(3) Since the number of fluorescent molecules is also known, the process control index is clarified, the sample processing process can be easily improved, and the accuracy can be easily increased.
(4) Further, since the sample itself can reduce the number of dummy samples added for reference, the cost can be easily reduced.

  Hereinafter, the light quantity measuring device of the present invention will be described with reference to the drawings.

FIG. 1 is a diagram for explaining the principle of a calibration method according to the present invention. In FIG. 1A, 1 is a power meter, 2 is an optical system, and 3 is an illumination reference light source such as an LED.
The power meter 1 is traceable to the national standard of optical power, and is compatible with the national standard. The power meter 1 measures the light quantity of the illumination reference light source 3 via the optical system 2, and values the light quantity of the illumination reference light source 3 based on the power meter 1.

  Next, as shown in FIG. 1B, the light quantity of the calibrated illumination reference light source 3 is detected by the light receiving element (not shown) of the light quantity detector (for example, camera) 4 through the optical system 2. . As a result, the relationship between the amount of light received by the light receiving element of the camera 4 and the output signal of the camera can be calibrated at a time.

  FIG. 2 shows an example of a calibration result obtained by such a calibration method. The figure shows the relationship between the absolute light quantity per unit area of the light receiving element and the gradation of the luminance signal output of the camera. When the light source 3 is green light, Y = 2.61E-06X + 2.57E-05, and when the light source 3 is red light, Y = 1.81E-06X-5.26E-05. In the expression, X is a gradation and Y is a light quantity.

  When the camera calibrated in this way is used in a light receiver of a biochip reader (for example, a biochip reader described in Japanese Patent Application Laid-Open No. 2001-31690 proposed by the applicant of the present application), the gradation of the measured image is measured. The amount of fluorescence can be directly measured from the value.

On the other hand, the amount of light generated from the fluorescent dye can be obtained by the following arithmetic processing.
The power ΔI of light absorbed by the fluorescent dye is given by the following equation.
ΔI = 2.3 × 103 × α × Io × n / (Na × S) [W] (1)
However,
α: Molar extinction coefficient 8 × 104 [M-1 cm-1]
Io: Incident light intensity [W]
n: number of molecules [pieces]
Na: Avogadro number 6 × 1023
S: Area [cm2]
It is.
By considering the quantum efficiency and the like, the amount of fluorescent light generated from the fluorescent dye can be estimated.

  Therefore, the number n of fluorescent molecules on the biochip can be directly estimated by associating the fluorescence light quantity measured using the camera calibrated by the calibration method with the estimated fluorescence light quantity value.

The present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.
For example, not only a biochip, but also general cells, dust (fluorescent dust) in a semiconductor process, fluorescent coatings such as a plasma panel, and the like can be measured.

  The light receiver is not limited to a so-called two-dimensional array in which the light receiving elements are arranged two-dimensionally, and a line sensor 4a in which the light receiving elements are arranged one-dimensionally as shown in FIG. 3 can also be used. It is. In this case, a two-dimensional image of the sample may be detected by adding a stage (not shown) and moving the sample 5 in a direction orthogonal to the arrangement direction of the line sensors 4a.

  Further, a light quantity amplification system such as an image intensifier may be inserted between the camera and the optical system. This makes it possible to measure faint light. In this case, it is necessary to perform calibration in a form including the image intensifier.

It is explanatory drawing for demonstrating the principle of the calibration method which concerns on this invention. It is a figure which shows an example of a calibration result. It is an Example figure at the time of using the light receiving element of a one-dimensional arrangement | sequence.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Power meter 2 Optical system 3 Reference light source for illumination 4 Light quantity detector 4a Line sensor 5 Sample

Claims (2)

Uses light emitted from a reference light source for illumination that has light receiving elements arranged in a one-dimensional or two-dimensional form and is measured by a power meter that is traceable to the national standard of optical power. And a light amount detector in which the relationship between the received light amount of each light receiving element and its output signal is calibrated at once,
A light quantity measuring device that measures the absolute light quantity of fluorescence generated from a biochip, a cell, a fluorescent coating, or fluorescent dust. Uses light emitted from a reference light source for illumination that has light receiving elements arranged in a one-dimensional or two-dimensional shape and is measured by a power meter that is traceable to the national standard of optical power. A light amount detector in which the relationship between the amount of light received by each light receiving element and its output signal is calibrated at once;
A means for calculating the number of molecules of the fluorescent object from the output signal of the light intensity detector that detects the amount of fluorescent light from the fluorescent object and the relational expression of the amount of light generated from the fluorescent dye;
A light quantity measuring apparatus for directly estimating the light quantity or the number of molecules or the quantity and number of molecules of the fluorescent object.

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