JP6357739B2 - Fluorescence detector - Google Patents

Description

  The present invention relates to a fluorescence detector provided in an automatic analyzer or the like.

  In a fluorescence detector provided in an automatic analyzer or the like, a band-pass interference filter having a specific wavelength band is provided to limit the wavelength of excitation light applied to the sample and detect specific fluorescence emitted from the sample. Is often used.

  The interference filter has the principle characteristic that the transmission spectrum shifts to the short wavelength side when the incidence of light on the filter surface is changed from normal incidence to oblique incidence. When an interference filter is used to select a fluorescence detection wavelength in the fluorescence detector, the transmission spectrum is shifted to the excitation light side (to the short wavelength side) by oblique incidence.

  When irradiating the sample with excitation light, the excitation light is scattered (diffusely reflected) in various directions from the container in which the sample is stored. When a part of the scattered excitation light is incident on the surface of the interference filter for detection obliquely, it passes through the interference filter whose transmission spectrum is shifted to the short wavelength side and is detected by the light receiving unit, thereby causing background noise. It becomes. When the background noise ratio increases, the change in the amount of fluorescence originally generated from the sample measured by the fluorescence detector is observed to be relatively small, so that the detection sensitivity decreases.

  As a method of reducing the amount of light incident obliquely on the surface of the interference filter, a method of providing a collimating lens, a diaphragm, or a color glass filter having wavelength selectivity for light absorption on the light incident side of the interference filter It has been known.

  The method of providing a collimating lens is that, as long as the sample cannot be regarded as a fluorescent point light source, the obliquely irradiated light remains. Therefore, measures are taken to make the volume of the sample small enough to be regarded as a point light source or to increase the optical system. There is a need. However, the former measure has a problem that the fluorescence intensity becomes low and detection becomes difficult, and the latter measure has a problem that leads to an increase in the size of the apparatus.

  As with the method of providing a collimating lens, the method of providing an aperture cannot provide a significant effect unless the sample can be regarded as a fluorescent point light source. Less.

  The colored glass filter is an optical filter having no dependency on the incident angle of the transmission spectrum, but there are few choices of the wavelength range that can be selected, and there is a problem that the color glass filter does not necessarily match the target optical system. Also, the color glass filter has a very slow transition from the stop band to the transmission band of the transmission spectrum as compared with the interference filter. Therefore, in preparation for the shift to the short wavelength side that occurs at the oblique incidence to the interference filter, the transmittance of the colored glass filter is 0% and the transmittance at the time of perpendicular incidence to the interference filter is 0%. Even if the color glass filter is selected so that the bands match, and the color glass filter is provided on the light incident side of the interference filter, the transmission band of the interference filter and the low transmittance band of the color glass filter overlap. However, there is a problem that the fluorescence detection sensitivity decreases.

  Therefore, an object of the present invention is a fluorescence detector comprising: excitation light irradiating means for irradiating a sample with excitation light; and fluorescence detecting means for detecting fluorescence at the light receiving unit by passing fluorescence emitted from the sample through an interference filter. An object of the present invention is to provide a fluorescence detector that can reduce the amount of interference light (excitation light) incident obliquely with respect to the surface of the interference filter and reaching the light receiving unit with a simple configuration.

  This invention made | formed in view of the said subject includes the following aspects.

That is, the first aspect of the present invention is:
A fluorescence detector comprising excitation light irradiation means for irradiating a sample with excitation light, and fluorescence detection means for detecting fluorescence emitted from the sample,
The fluorescence detection means includes an opening and a closing part, and a cylindrical body whose inner wall is shielded from light, a collimating lens provided on the opening part side, a light receiving part provided on the closing part side, and the collimating lens, The fluorescence detector having an interference filter provided on an optical axis connecting to the light receiving unit, and having a distance from the collimating lens to the light receiving unit being longer than an effective diameter of the collimating lens.

  Moreover, the 2nd aspect of this invention is a fluorescence detector as described in said 1st aspect which provided the unevenness | corrugation in the inner wall of a cylindrical body.

  The third aspect of the present invention is the second aspect, wherein the cylindrical body is a cylindrical shape, and the unevenness provided on the inner wall of the cylindrical body is a spiral groove formed along the inner wall. It is a fluorescence detector as described in an aspect.

  Hereinafter, the present invention will be described in detail.

  The fluorescence detector of the present invention comprises a fluorescence detection means constituting the fluorescence detector, a cylindrical body provided with an opening and a closing portion and whose inner wall is shielded from light, a collimating lens provided in the opening, and the closing portion. A light receiving portion provided on the optical axis, and an interference filter provided on an optical axis connecting the collimating lens and the light receiving portion, and the distance from the collimating lens to the light receiving portion is effective for the collimating lens. It is characterized by being longer than the diameter.

  In the fluorescence detector of the present invention, the interference filter may be provided at an arbitrary position as long as it is on the optical axis connecting the collimating lens and the light receiving unit. As an example, when an interference filter is provided so as to overlap the light receiving part side of the collimating lens, the collimating lens generally has the effect of condensing the light incident from the opening of the cylindrical body on the light receiving part provided at the closed part of the cylindrical body. Have. However, since the sample that emits fluorescence and the container that accommodates it have a certain volume, if the sample and the container are arranged in the vicinity of the collimating lens, the field volume from the collimating lens becomes large. Even if the light passes through the collimating lens, it cannot block light incident obliquely on the surface of the interference filter (obliquely incident light). Obliquely incident light reaching the light receiving portion can be prevented as the effective diameter of the collimating lens is smaller and as the distance between the collimating lens and the light receiving portion is larger. However, simply reducing the effective diameter of the collimating lens decreases the sensitivity of the fluorescence detector, and simply increasing the distance between the collimating lens and the light receiving portion increases the size of the fluorescence detector. Therefore, in the fluorescence detector of the present invention, the distance from the collimating lens provided at the opening of the cylindrical body to the light receiving portion provided at the closed portion of the cylindrical body is set longer than the effective diameter of the collimating lens. This prevents the incident light from directly reaching the light receiving section and prevents the fluorescence detector from becoming large. When the interference filter is provided in the vicinity of the light receiving part, the light that is obliquely transmitted through the collimating lens provided in the opening of the cylindrical body (obliquely incident light) is light-shielded before reaching the light receiving part. Since the obliquely incident light itself that is mostly absorbed by the inner wall and reaches the interference filter is extremely small, it is possible to prevent the obliquely incident light from reaching the light receiving section as a result.

The transmission peak wavelength λ when obliquely incident at an incident angle φ through an interference filter having a transmission peak wavelength of λ _max when perpendicularly incident can be calculated by the following equation.

λ = λ _max √ (1- [sin φ / n] ² )
λ: transmission peak wavelength at oblique incidence λ _max : transmission peak wavelength at perpendicular incidence n: relative refractive index (n = n ₀ (refractive index of air) / _ne (effective refractive index of interference film))
φ: Incident angle For example, when a bandpass filter having a relative refractive index of 1.8 and a peak wavelength of 500 nm is used as an interference filter, when light is incident obliquely at an incident angle of 30 °, the peak wavelength becomes 480 nm, and the transmission band Shifts by 20 nm toward the short wavelength side. Therefore, when a band-pass filter having a peak wavelength of 500 nm is provided as an interference filter for detection on the light receiving part side of the collimator lens, and a band-pass filter having a center wavelength of 480 nm is used as the excitation light interference filter, the detection interference filter is set to the incident angle What is necessary is just to set the distance from the collimating lens provided in the opening part of the cylindrical body to the light receiving part so that the excitation light obliquely incident at 30 ° does not directly reach the light receiving part.

  Specifically, when the distance from the collimating lens to the light receiving unit is made equal to the effective diameter of the collimating lens, the maximum incident angle of light directly incident on the light receiving unit is about 27 °, and therefore the fluorescence detector of the present invention. If so, it can be seen that the excitation light obliquely incident at the aforementioned incident angle of 30 ° can be prevented from being directly incident on the light receiving portion. If an increase in the size of the fluorescence detector is permitted, it is more preferable that the distance from the collimating lens to the light receiving unit is at least twice the effective diameter of the collimating lens.

  For light that is obliquely incident on the interference filter at a angle greater than or equal to the maximum incident angle of light that is directly incident on the light receiving unit, the transmission band of the interference filter shifts to a shorter wavelength side. In the fluorescence detector of the present invention, since the inner wall of the cylindrical body constituting the optical waveguide from the interference filter to the light receiving part is subjected to a light shielding process (for example, a black process or a non-reflective process), excitation light that passes through the interference filter Is absorbed by the inner wall of the cylindrical body. However, even if the inner wall of the cylindrical body that constitutes the optical waveguide from the interference filter to the light receiving part is shielded, not all of the excitation light that passes through the interference filter is absorbed, but is reflected by the inner wall and received. There is also a small amount of light reaching the part. Therefore, if irregularities are periodically provided on the inner wall of the cylindrical body, the excitation light reflected by the irregularities on the inner wall can be reflected to the opening side of the cylindrical body, and the amount of excitation light reaching the light receiving section can be further reduced. Therefore, it is preferable. In addition, when the unevenness | corrugation provided in the inner wall of a cylindrical body is made into the rectangular uneven | corrugated shape parallel to the light which permeate | transmitted the collimating lens (FIG. 2), the ratio of a convex part is so good that a concave part is deep. Moreover, when the uneven | corrugated cross section provided in the inner wall of a cylindrical body is made into the shape of a triangle (sawtooth shape, bellows shape) (FIG. 3), the angle of a peak and a valley is so preferable that it is small (sharp). Further, it is more preferable in terms of workability and cost that the irregularities are formed into a female screw shape (when a spiral groove is formed along the inner wall).

  The shape of the cylindrical body is not particularly limited, and examples thereof include a cylindrical shape, a spherical shape, and a truncated cone shape having a closed portion (light receiving portion) side having a diameter larger than the effective diameter of the collimating lens.

  The fluorescence detector of the present invention includes an excitation light irradiating means for irradiating a sample with excitation light, a cylindrical body provided with an opening and a closing portion and having an inner wall shielded, a collimator lens provided in the opening, and the closing portion. A fluorescence detecting means for detecting fluorescence emitted from the sample, and a light detecting portion provided on the optical axis connecting the light receiving portion to the collimating lens, and from the collimating lens The distance to the light receiving part is longer than the effective diameter of the collimating lens. The fluorescence detector of the present invention can reduce the irradiation amount of the excitation light transmitted through the interference filter to the light receiving unit by being obliquely incident on the surface of the interference filter. In addition, the fluorescence detector of the present invention is effective by increasing the distance from the collimating lens provided in the opening of the cylindrical body to the light receiving section, that is, by substantially increasing the length of the cylindrical body. Compared to a method (a method of providing a diaphragm or a colored glass filter), interference light caused by excitation light can be reduced with a simple configuration.

  If the inner wall of the cylindrical body is provided with irregularities, the oblique incident light before or after passing through the collimating lens and passing through the interference filter can be reflected to the opening side of the cylindrical body by the irregularities. This is preferable because irradiation of the light receiving part can be further reduced.

It is a block diagram of the fluorescence detector of the present invention. It is a figure which shows the one aspect | mode of the fluorescence detector of this invention. It is a figure which shows the one aspect | mode of the fluorescence detector of this invention. It is a figure which shows the result of Example 1.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the said Example.

Example 1
Using the fluorescence detector shown in FIG. 1, the amount of light irradiated to the light receiving unit 23 when the distance from the collimating lens 21 to the light receiving unit 23 was changed was measured. The excitation light source 11 is a light emitting diode of 470 nm, the excitation light interference filter 12 is a transmission band 465 nm to 475 nm, the detection interference filter 22 is a transmission band 505 nm to 515 nm, a relative refractive index of 1.8, an inner diameter A φ8 mm bandpass filter is used as the sample container 31, a colorless transparent polypropylene container with a tip curvature of 2 mm, and a cylindrical body 20 covering the optical waveguide from the detection interference filter 22 to the light receiving unit 23, and the inner wall surface is black chrome. Each of the treated hollow tubes having an inner diameter of φ8 mm was used. The detection interference filter 22 is provided so as to overlap the light receiving portion 23 side of the collimating lens 21, and the distance between the tip of the sample container 31 and the collimating lens 21 is 8 mm.

  The amount of fluorescence emitted from the fluorescent sample 32 including the nucleic acid probe modified with the fluorescent dye and the blank (water not including the fluorescent sample) after changing the distance from the collimating lens 21 to the light receiving unit 23 in the range of 5 mm to 17 mm. Was measured. The results are shown in FIG. 4 (black circle: fluorescent sample, black square: blank, x: fluorescent sample / blank ratio). When the distance from the collimating lens 21 to the light receiving unit 23 is shorter than the effective diameter (φ8 mm) of the collimating lens 21, an increase in detector output is confirmed for both the fluorescent sample and the blank, and the S / N ratio (fluorescent sample / blank ratio). Decreased significantly. On the other hand, when the distance from the collimating lens 21 to the light receiving unit 23 is made longer than the effective diameter (φ8 mm) of the collimating lens 21, an increase in detector output is suppressed for both the fluorescent sample and the blank, and the S / N ratio is improved. I understand that. In other words, by making the distance from the collimating lens 21 to the light receiving unit 23 longer than the effective diameter of the collimating lens 21, interference caused by excitation light that is incident on the light receiving unit obliquely with respect to the surface of the interference filter. It can be seen that light can be reduced.

Example 2
Using the fluorescence detector shown in FIG. 1 and the fluorescence detector shown in FIG. 3, the effect of providing irregularities on the inner wall of a cylindrical body as an optical waveguide was verified.

  A 450 nm light emitting diode as the excitation light source 11, a bandpass filter with a transmission band of 445 nm to 455 nm as the interference filter 12 for excitation light, a transmission band of 470 nm to 495 nm as the interference filter 22 for detection, a relative refractive index of 1.8, and an inner diameter of φ8 mm. A Buntpass filter having a cylindrical body 20 with an inner wall surface of black chrome-treated hollow tube with an inner diameter of φ8 mm and an M8 triangular female screw (pitch 1.25 mm, valley diameter 8 mm) equivalent unevenness 25 provided on the inner wall The distance from the collimating lens 21 to the light receiving unit 23 was 13 mm. The effective diameter of the collimating lens 21, the used sample container 31, and the distance from the tip of the sample container 31 to the collimating lens 21 are the same as those in the first embodiment.

  Using the fluorescence detector shown in FIG. 1 and the fluorescence detector shown in FIG. 3, the amount of fluorescence emitted from a fluorescent sample 32 containing a nucleic acid probe modified with a fluorescent dye and a blank (water not containing a fluorescent sample) was measured. The results are shown in Table 1. It can be seen that by providing irregularities on the inner wall of the cylindrical body as the optical waveguide, the detector output emitted from the blank is significantly reduced, and as a result, the S / N ratio (fluorescent sample / blank ratio) is improved.

DESCRIPTION OF SYMBOLS 11 Excitation light source 12 Excitation light interference filter 20 Tubular body (optical waveguide)
DESCRIPTION OF SYMBOLS 21 Collimating lens 22 Interference filter for detection 23 Light-receiving part 24 Rectangular unevenness 25 Female screw-shaped unevenness 31 Sample container 32 Fluorescent sample

Claims (3)

A fluorescence detector comprising excitation light irradiation means for irradiating a sample with excitation light, and fluorescence detection means for detecting fluorescence emitted from the sample,
The fluorescence detection means includes an opening and a closing part, and a cylindrical body whose inner wall is shielded from light, a collimating lens provided on the opening part side, a light receiving part provided on the closing part side, and the collimating lens, consists interference filter provided on an optical axis connecting the light receiving portion, and shorter than twice the length rather effective diameter than the effective diameter of the distance from the collimating lens to said light receiving portion is the collimating lens, The fluorescence detector. The fluorescence detector according to claim 1, wherein irregularities are provided on the inner wall of the cylindrical body. The fluorescence detector according to claim 2, wherein the cylindrical body is cylindrical, and the unevenness provided on the inner wall of the cylindrical body is a spiral groove formed along the inner wall.

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