JP2010190713A - Fluorescence measuring apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence measuring apparatus for performing fluorescence measurement of a minute amount of a liquid sample wherein the reproducibility is high, the sample is easily held, and the measurement accuracy is high. <P>SOLUTION: The fluorescence measuring apparatus is achieved by the following configurations: in a first configuration, a liquid sample disposed at an excitation light converging position of an epi-illumination optical system object lens using a fluorescent cube is held in a hole of a sample holding plate wherein the hole passes through the plate perpendicular to the optical axis; in a second configuration, an aqueous solution sample is held with an affinity in a hydrophilic circular area on the sample holding plate and the circumference of the circular area is rendered water repellent, and thus the aqueous solution sample is bound within the hydrophilic circular area; and in a third configuration, droplets of the liquid sample is made to fly. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a fluorescence measuring apparatus for measuring the fluorescence of a small amount of liquid sample, and particularly to a sample holding method during measurement.

  Measuring the fluorescence of a liquid sample in the microliter order is highly useful when the amount of a substance such as a fluorescently labeled nucleic acid is slightly restricted.

  In the prior art, the sample is held by surface tension between two surface anvils that are relatively operable and substantially parallel, and the optical path is established between the respective wet areas of the two surfaces. An apparatus is disclosed in Japanese translations of PCT publication No. 2008-530554.

  However, this method has the following problems. First, the two anvils that hold the sample require a complicated and precise structure because they require opening and closing operations. Secondly, in addition to the anvil opening / closing operation, two anvils need to be cleaned for measurement, but it is not easy to clean a fine and complicated structure, and it is troublesome and residue is likely to remain.

Special table 2008-530554

  The problem to be solved is to realize a fluorescence measuring apparatus that can easily hold a small amount of liquid sample with good reproducibility and can perform fluorescence measurement with high accuracy.

  The means of the present invention for solving the above-described problem is that a liquid sample disposed at an excitation light condensing position of an objective lens of an epi-illumination optical system using a fluorescent cube is provided with a hole penetrating perpendicularly to the optical axis in the first configuration. In the second configuration, the aqueous solution sample is held in the hydrophilic circular region on the sample holding plate by affinity, and the periphery of the circular region is made water-repellent by holding the sample in the hole of the sample holding plate. In a third configuration by flying liquid sample drops.

  In the first configuration, the fluorescence measuring apparatus according to the present invention has a second configuration in which a liquid sample is injected into a through hole of a sample holding plate with a pipette and measurement is performed, and then a cleaning liquid is poured around the hole and wiped off. In the third configuration, the aqueous sample is dropped onto the hydrophilic circular area on the sample holding plate and measured, and then the circular area is wiped with water. In the third configuration, the liquid sample is dropped with the pipette to measure. Since the measurement work is completed by collecting the samples that have been dropped after being performed, a fluorescence measuring apparatus having a simple structure and simple operation can be realized in any case.

It is explanatory drawing which showed the whole fluorescence measuring device. (Example 1) It is explanatory drawing which showed the sample holding plate of the fluorescence measuring device. (Example 1) It is explanatory drawing which showed the sample holding plate of the fluorescence measuring device. (Example 2) It is explanatory drawing which showed the sample holding plate of the fluorescence measuring device. (Example 3) It is explanatory drawing which showed the whole fluorescence measuring device. Example 4

  In the first embodiment, the sample holding plate is arranged so that the hole of the sample holding plate having a hole penetrating perpendicularly to the optical axis coincides with the excitation light condensing position of the objective lens of the epi-illumination optical system using the fluorescent cube. It is arranged.

  In the second embodiment, the sample holding plate is arranged so that the hydrophilic circular area on the sample holding plate coincides with the excitation light condensing position of the objective lens of the epi-illumination optical system using the fluorescent cube.

  In the third embodiment, the epi-illumination optical system is tilted horizontally so that the liquid sample dropped vertically from the pipette passes through the excitation light condensing position of the objective lens of the epi-illumination optical system using the fluorescent cube. A means for arranging and fixing the pipette is provided immediately above the excitation light condensing position.

  FIG. 1 shows an embodiment of the first mode of the apparatus of the present invention. Reference numeral 1 is an excitation light source, and 2 is a basic optical system for fluorescence detection, which is called a fluorescence cube. The fluorescent cube 2 includes an excitation filter 3 that is a narrow band interference filter that selectively transmits an excitation wavelength, a dichroic beam splitter 4 that reflects the excitation wavelength and transmits the fluorescence wavelength, and transmits the fluorescence wavelength and reflects the excitation wavelength. The absorption filter 5 that is an interference filter is combined and integrated. However, in the present invention, being integrated is not a necessary requirement, and these combinations including the case where they are individually installed are called fluorescent cubes. The excitation light 6 emitted from the excitation light source is wavelength-purified by the excitation filter 3, reflected by the dichroic beam splitter 4, and condensed at the excitation light condensing position by the objective lens 7. The sample held on the sample holding plate 8 is arranged at the excitation light condensing position. The fluorescence 9 emitted from the sample that has absorbed the excitation light is collimated by the objective lens 7, passes through the dichroic beam splitter 4, and is received by the photodetector 11 by the condenser lens 10. If the photodetector 11 is a spectroscopic detector including a monochromator, it can be a spectrofluorometric apparatus. The above optical system is known as an epi-illumination optical system. Reference numeral 12 denotes excitation light transmitted through the sample.

  The present invention basically uses an epi-illumination optical system, and since the feature is in the sample holding plate portion, it will be described in detail with reference to FIG. In FIG. 2, (a) has a hole 13 penetrating parallel to the optical axis of the excitation light irradiated in the perspective view of the sample holding plate 8. (B) shows the cross section, and 14 is a liquid sample. When the fixed amount is dropped onto the hole 13 with a pipette, the liquid sample 14 gets wet on the inner surface of the hole and is held in a certain shape by the surface tension. Since the sample holding plate is arranged so that the hole 13 coincides with the excitation light condensing position of the objective lens 7, the excitation light irradiates the liquid sample 14 in a concentrated manner, and fluorescence is generated. In general, the property of the generated fluorescence varies as the shape of the sample changes. However, since a certain shape can be obtained as described above, measurement variation is small and measurement with good reproducibility is possible. The shapes of the holes 13 do not need to be parallel, and for example, when the tapered holes are formed along the excitation light profile as shown in (c), an additional effect is obtained such that the liquid sample 14 can be excited efficiently. . The amount of sample that can be held can be controlled by the plate thickness and hole diameter, so that approximately the same amount of sample as the hole volume can be held. A suitable liquid sample of 1 microliter has a thickness of about 1.5 mm and a hole diameter of about 1 mm. In addition, it is possible to provide two or more holes in one sample holding plate 8, and in this case, a means for positioning all the holes at the excitation light condensing position is required, but the mechanism is various and easy. Omitted.

In the present embodiment, the method for supplying the liquid sample has been described above. In the cleaning after the measurement, after wiping the sample, the hole 13 and the periphery thereof are wiped with a cleaning liquid corresponding to the sample. Since the structure is simple, sample supply and cleaning can be performed easily and reliably.

  FIG. 3 shows an embodiment of the second aspect of the apparatus of the present invention. Except for the sample holding plate portion, it is the same as the embodiment of the first embodiment, and therefore, the overall view is omitted, and an enlarged main portion is shown. In FIG. 3, (a) is a perspective view of the sample holding plate 15 in which the circular region 16 is made hydrophilic and its periphery is made water repellent. (B) shows the cross section, and 17 is an aqueous solution sample. Since the circular region 16 is hydrophilic, it has good wettability with respect to an aqueous solution, and since the periphery is water-repellent, the bottom area of the sample is constrained by the circular region. Is done. Since the sample holding plate is disposed so that the circular area 16 coincides with the excitation light condensing position of the objective lens 7, the excitation light irradiates the aqueous solution sample 17 in a concentrated manner, and fluorescence is generated. The amount of sample that can be held can be controlled by the area of the circular region. As in the case of the first embodiment, it is possible to provide two or more circular regions on one sample holding plate 15. It is the same that the variation in measurement is small because the sample shape is constant.

  In the present embodiment, the method for supplying the liquid sample has been described above. In the cleaning after the measurement, the periphery of the circular region 16 is wiped with a cleaning liquid corresponding to the sample after wiping the sample. Since the structure is simple, sample supply and cleaning can be performed easily and reliably.

  FIG. 4 shows another embodiment of the second aspect of the apparatus of the present invention. This example is basically the same as the first embodiment except for the portion of the sample holding plate 18, but the top and bottom are inverted and excitation light is irradiated from below the sample holding plate 18 to detect fluorescence. In the figure, 19 is an aqueous solution sample. In this example, the sample holding plate 18 needs to be transparent to excitation light and fluorescence, and glass, quartz glass, transparent acrylic resin, or the like can be used. The advantage of this example is that the sample holding plate 18 is located at the uppermost position, so that it is easy to supply and clean the sample from the upper surface of the apparatus.

  FIG. 5 shows an embodiment of the third mode of the apparatus of the present invention. This example is basically the same as the example of the first embodiment except for the portion where the sample is interposed, but the optical system is turned over and arranged. That is, excitation light is irradiated horizontally to detect fluorescence. In this example, the liquid sample dropped from the pipette is dropped through the excitation light condensing position of the objective lens 7. That is, the fluorescence emitted when the sample droplet that makes a drop motion passes through the excitation light condensing position is detected. In the case of this example, the sample holding means itself is not required during the measurement, and only the container 19 for collecting and collecting the dropped sample is required. However, a guide 20 for positioning the pipette is provided to allow the sample droplet to pass through the excitation light condensing position accurately.

In this embodiment, since there is no sample holding portion, there is no need for cleaning.

INDUSTRIAL APPLICABILITY The present invention can be used in the bio field such as gene analysis that requires fluorescence measurement of a small amount of liquid sample and the medical field such as virus detection.

DESCRIPTION OF SYMBOLS 1 Excitation light source 2 Fluorescence cube 3 Excitation filter 4 Dichroic beam splitter 5 Absorption filter 6 Excitation light 7 Objective lens 8 Sample holding plate 9 Fluorescence 10 Condensing lens 11 Photo detector 12 Excitation light which permeate | transmitted sample 13 Hole 14 Liquid sample 15 Sample Holding plate 16 Hydrophilic circular region 17 Aqueous sample 18 Sample holding plate 19 Aqueous sample 20 Sample collection container 21 Pipette positioning guide

Claims (3)

  A liquid sample arranged at an excitation light condensing position of an objective lens of an epi-illumination optical system using a fluorescent cube is held in the hole of a sample holding plate having a hole penetrating in parallel to the optical axis. Fluorescence measuring device.   An aqueous solution sample placed at the excitation light condensing position of the objective lens of the epi-illumination optical system using a fluorescent cube is held by affinity in the hydrophilic circular area on the sample holding plate, and the periphery of the circular area is made water-repellent By so doing, an aqueous solution sample is constrained within a hydrophilic circular region. A fluorescence measuring apparatus, wherein a liquid sample droplet is caused to fly to an excitation light condensing position of an objective lens of an epi-illumination optical system using a fluorescent cube.