JP2004325174A - Fluorescence detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence detector which efficiently emits sufficient exciting light without any irregularities and reliably detects fluorescence. <P>SOLUTION: The fluorescence detector comprises: a sample base 1 on which a sample 202 is placed; an LED light source unit 11 that emits light to the sample 202 and excites a fluorescent substance contained in the sample 202; and a fluorescence detection means having an objective lens 5 for condensing fluorescence generated by exciting a fluorescent coloring matter substance by the exciting light, a filter 22 for selectively transmitting the fluorescence generated by exciting the fluorescent substance by the exciting light, and a photodetector 24 for detecting the fluorescence passing through the objective lens 5. The sample base 1 and the LED light source unit 11 are relatively, movably provided and light from the LED light source unit 11 is emitted to the sample 202 concurrently with the relative movement. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fluorescence detection device that detects the expression level of a gene, the presence or absence of a mutation, and the like using fluorescence.
[0002]
[Prior art]
2. Description of the Related Art Conventionally, the main methods of gene testing that have been put to practical use include extracting nucleic acids from a biological sample, and using PCR (Polymerase Chain Reaction) or NASBA (Nucleic Acid Sequence-Based Amplification): based on nucleic acid sequences. Amplification method) is used to amplify a target gene by amplifying a nucleic acid labeled with a radioisotope or a fluorescent dye, and measure the base sequence of the target gene or its concentration. .
[0003]
In addition, electrophoresis is used for inspection of gene expression levels and analysis of mutations and the like. However, this electrophoresis method has problems that it takes time and effort for the measurement, and there is a limit to the measurement that can be performed at one time.
[0004]
Therefore, recently, capillary electrophoresis, which allows a fluorescently labeled nucleic acid to react in a plurality of capillaries and rapidly process many samples at once, has been widely used. According to this capillary electrophoresis method, measurement can be performed in a shorter time and more easily than in a method using a conventional electrophoresis method.
[0005]
Furthermore, recently, a testing method using a DNA chip capable of simultaneously performing a plurality of gene tests has been newly developed. Here, a DNA chip is one in which a large number of DNA probes are immobilized on the surface of a glass substrate, or one in which a large number of oligo probes synthesized on a minute area on a silicon wafer by applying a semiconductor manufacturing process are attached. is there.
[0006]
In such an inspection method using a DNA chip, a plurality of base sequences and expression levels of DNA in a sample can be simultaneously determined. Also, various tests such as analysis of a large amount of gene expression and a plurality of mutations can be performed by applying a DNA chip. Further, from data obtained using a DNA chip, many genes can be classified into a plurality of groups, and information on gene fluctuations associated with development and differentiation can be obtained.
[0007]
However, although the gene analysis method using a DNA chip has the advantage that a large number of tests can be performed at one time, it requires a long test time, has many test steps throughout, and requires complicated operations. There is a problem that it is difficult to obtain good detection results.
[0008]
In order to overcome such a problem, a method using a porous filter as a carrier of a DNA chip, which has good reproducibility and can perform the same test as a DNA chip in a short time, or a hybridization reaction (Patent Document 1, Patent Document 2, and Patent Document 3) have been developed.
[0009]
Further, recently, a genetic test apparatus has been proposed which performs a gene analysis using a DNA microarray and fluorescence emitted from a reaction product generated in a reaction vessel of the DNA microarray. This device is based on a microscope, and causes a hybridization reaction between a target nucleic acid and a nucleic acid probe that have been fluorescently labeled in advance in a reaction vessel of a DNA microarray, and the reaction is captured by the DNA microarray. A fluorescent signal is obtained from the fluorescent substance, and a fluorescent image is obtained based on the fluorescent signal. Here, the DNA microarray has a structure in which a number of small reaction vessels (liquid storage sections) for storing a sample are arranged.
[0010]
In such a genetic test method using a DNA microarray, it is necessary to efficiently excite a fluorescent substance to generate stable fluorescence from a reaction product generated in a reaction vessel of the DNA microarray. Is extremely important in doing
[0011]
2. Description of the Related Art Conventionally, an LED (Light Emitting Diode) excitation light source is used, as disclosed in Patent Document 4, for example, as disclosed in Patent Document 4 by irradiating a fluorescent substance with excitation light from a light source. The fluorescent substance is excited by the emitted excitation light, and a fluorescent signal is received by the solid-state imaging device through filter means for selectively transmitting the wavelength of the incident light with respect to the fluorescent light emitted by the excited fluorescent substance, In some cases, a fluorescent image is obtained based on the fluorescent signal.
[0012]
Further, as disclosed in Patent Document 5, light from an LED is irradiated to a well as excitation light via a dichroic mirror and an objective lens, and fluorescence emitted from a sample is emitted from a pinhole unit via an objective lens and a dichroic mirror. There is also a device in which detection is performed by a photomultiplier tube via a photomultiplier.
[0013]
Further, as disclosed in Patent Document 6, semiconductor light sources are two-dimensionally arranged to illuminate a sample via a condenser lens as a surface light source, and light transmitted through the sample is made incident on an objective lens. That is, the illumination light from the two-dimensionally arranged semiconductor light sources is sent along the optical axis, and is incident on the objective lens in a state where it is aligned with the optical axis of the objective lens so that an enlarged image can be obtained. There is something.
[0014]
Furthermore, as disclosed in Patent Document 6, a semiconductor excitation light source is used to irradiate a sample with light through an objective lens to excite a fluorescent dye inside the sample, and to fluoresce the sample with the same optical power as incident light. Some are detected by a photodetector through the system.
[0015]
[Patent Document 1]
Japanese Patent Publication No. 9-504864
[0016]
[Patent Document 2]
JP-T-2000-515251
[0017]
[Patent Document 3]
Japanese Patent Publication No. 2001-501301
[0018]
[Patent Document 4]
JP-A-10-132744
[0019]
[Patent Document 5]
JP-A-2002-116148
[0020]
[Patent Document 6]
Japanese Patent Publication No. 7-122694
[0021]
[Patent Document 7]
US Pat. No. 6,154,282
[0022]
[Problems to be solved by the invention]
By the way, usually, the LED light source does not have a sufficient light intensity to excite the fluorescent substance, and cannot generate sufficient fluorescence from the fluorescent substance. For this reason, it is often difficult to receive light with commonly used light detecting means such as a CCD camera. When using an LED light source as a light source for exciting a fluorescent substance, select a model with the highest output intensity as much as possible. Needless to say, it is necessary to devise a method of bundling two or more LEDs, bringing them as close to the sample surface as possible, so that a sufficient excitation light is uniformly applied to the fluorescent substance.
[0023]
However, Patent Document 4 does not specifically describe a method for effectively irradiating the fluorescent substance with the excitation light from the LED light source in consideration of the above. One of the LED light sources is disposed directly below the fluorescent substance, and the optical axis of the excitation light from the LED light source and the optical axis of the camera lens coincide with each other. This means that part of the excitation light that has passed through the fluorescent substance travels along the same optical path as the fluorescent light emitted from the fluorescent substance. This causes a problem of lowering.
[0024]
In each of Patent Literature 5, Patent Literature 6, and Patent Literature 7, the optical axis of the illumination light from the semiconductor light source coincides with the optical axis of the objective lens. For this reason, although the respective optical paths of the excitation light and the fluorescence signal light passing through the same optical path are separated by the dichroic mirror, a part of the excitation light passes through the dichroic mirror and passes through the optical path through which the fluorescence emitted from the fluorescent substance passes. It may be mixed and become noise light. In addition, since it is technically impossible for a dichroic mirror to separate the reflection and transmission of light of a desired wavelength with 100% efficiency, the signal light intensity due to the reflection, scattering, transmission and absorption of desired fluorescence Cannot be avoided. Further, two optical paths of the excitation light and the fluorescent light emitted from the fluorescent substance must be accurately matched, and there is a problem that adjustment of the optical axis requires time and effort.
[0025]
The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a fluorescence detection device that can efficiently irradiate excitation light without unevenness and perform highly reliable fluorescence detection.
[0026]
[Means for Solving the Problems]
According to the first aspect of the present invention, there is provided a sample stage on which a sample is placed, a semiconductor light source means for irradiating the sample with light and exciting a fluorescent substance contained in the sample, and a fluorescent substance is excited by at least the excitation light. A condensing lens for condensing the emitted and emitted fluorescent light, a filter for exciting the fluorescent substance excited by the excitation light and selectively transmitting the emitted fluorescent light, and a light for detecting the fluorescent light passing through the condensing lens A fluorescence detector having a detector, wherein the sample stage and the semiconductor light source are provided so as to be relatively movable, and simultaneously with the relative movement, light from the semiconductor light source is supplied to the sample. Irradiation is performed.
[0027]
According to a second aspect of the present invention, in the first aspect of the present invention, the semiconductor light source means includes an LED light source that emits excitation light and light from the LED light source that is disposed on an optical path of light emitted from the LED light source. , A filter, a diffusion plate, a lens, or all of them, which selectively transmit the above wavelengths.
[0028]
According to a third aspect of the present invention, in the first or second aspect, a plurality of the semiconductor light source units are provided so as to rotate or reciprocate along the periphery of the condenser lens.
[0029]
According to a fourth aspect of the present invention, in the first or second aspect of the present invention, the plurality of semiconductor light sources are provided so as to be rotatable or reciprocable along the periphery of the condenser lens, and the number of times of rotation or reciprocation is an integer. It is characterized by being performed.
[0030]
According to a fifth aspect of the present invention, in the third or fourth aspect, the irradiation point of light from the semiconductor light source means to the sample is set such that an irradiation point of a peripheral portion of the sample is an irradiation point of a central portion of the sample. It is characterized in that it is set more densely than.
[0031]
The invention according to claim 6 is characterized in that, in the invention according to any one of claims 3 to 5, the semiconductor light source means has light emission wavelengths having substantially the same spectral characteristics.
[0032]
The invention according to claim 7 is characterized in that, in the invention according to any one of claims 3 to 5, the semiconductor light source means has different spectral characteristics of emission wavelength.
[0033]
The invention according to claim 8 is the invention according to any one of claims 1 to 7, wherein the semiconductor light source means is provided around the condenser lens so as to be rotatable around an optical axis of the condenser lens. Are held along the periphery of the condenser lens, and the semiconductor light source means is set at a predetermined angle with respect to the optical axis of the condenser lens so that light emitted from the semiconductor light source means is irradiated on the sample. A light source holding member that holds the semiconductor light source at an angle, and a light source holding member that is provided at a holding portion of the semiconductor light source means of the light source holding member; And a position adjusting means for adjusting at least one of the inclination directions.
[0034]
According to a ninth aspect of the present invention, in the first aspect of the present invention, there is provided a driving unit for supplying a driving current to the semiconductor light source unit, and the light source holding member is driven by the driving unit. It is characterized by having power supply means for supplying a current to the semiconductor light source means.
[0035]
According to a tenth aspect of the present invention, in the first aspect of the present invention, the photodetector includes an imaging unit, and further includes a position detection unit that detects a rotational position of the light source holding member. The exposure time optimal for the light detection of the imaging means is determined based on the output of the detection means.
[0036]
According to an eleventh aspect of the present invention, in the invention according to any one of the first to tenth aspects, further, the light intensity detecting means for detecting the intensity of the excitation light to the sample near the sample and the semiconductor light source means are driven. A driving means for supplying a driving current to the semiconductor light source means, the driving means having a controllable magnitude of the driving current, based on the intensity of the excitation light detected by the light intensity detecting means. It is characterized in that the drive current can be controlled.
[0037]
According to a twelfth aspect of the present invention, in the invention according to any one of the first to tenth aspects, further, the light intensity detecting means for detecting the intensity of the excitation light to the sample near the sample and the semiconductor light source means are driven. A driving means for supplying a driving current to the semiconductor light source means, the driving means having a controllable magnitude of the driving current, based on the intensity of the excitation light detected by the light intensity detecting means. It is characterized in that the drive current can be individually controlled.
[0038]
As a result, according to the present invention, the sample stage and the semiconductor light source means are provided so as to be relatively movable, and with this relative movement, the light from the semiconductor light source means is irradiated on the sample. Excitation light having an intensity sufficient to excite the fluorescent substance can be efficiently obtained.
[0039]
Further, according to the present invention, the irradiation point of the light from the semiconductor light source means on the sample surface is set densely at the peripheral portion of the sample as compared with the irradiation point at the central portion. When the sample rotates around the objective lens, the entire surface of the sample can be excited uniformly and evenly.
[0040]
Further, according to the present invention, the semiconductor light source does not need to simultaneously irradiate the entire surface of the sample with light, and the semiconductor light source rotates the periphery of the objective lens to irradiate the entire surface of the sample with light. Therefore, the entire surface of the sample can be uniformly and efficiently excited by the minimum number of semiconductor light sources.
[0041]
Furthermore, according to the present invention, since the optical path for the excitation light through which the excitation light passes and the optical path of the fluorescence detecting means having the condensing lens are separated, an optical element such as a dichroic mirror for attenuating the light can be omitted, and the light can be detected. Light intensity loss can be minimized.
[0042]
Furthermore, according to the present invention, since the direction of the light emitted from the semiconductor light source means and the direction of inclination of the condenser lens with respect to the optical axis can be adjusted, light can be condensed at a desired specific portion of the sample. Thus, efficient excitation of the fluorescent substance can be performed.
[0043]
Further, according to the present invention, the drive current to the semiconductor light source means can be individually controlled based on the light intensity obtained near the sample, so that the light from the semiconductor light source means can be adjusted to the same brightness, Can be illuminated with uniform brightness.
[0044]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0045]
(First Embodiment)
FIG. 1 shows a schematic configuration of a fluorescence inspection apparatus based on a microscope structure to which the present invention is applied.
[0046]
In FIG. 1, reference numeral 1 denotes a sample stage, on which a DNA reaction container 2 is mounted as a subject.
[0047]
Here, FIG. 2 shows a DNA slide glass reaction container 3 as a specific example of the DNA reaction container 2. The DNA slide glass reaction vessel 3 has a large number of minute sample vessels 301 arranged in a slide glass-like reaction vessel, and a DNA microarray 302 is laid in these sample vessels 301. Then, when a sample solution (test sample) is injected into the sample tank 301, a hybridization reaction occurs between the target nucleic acid and the nucleic acid probe that have been fluorescently labeled in advance in the DNA microarray 302. Fluorescence is emitted from a captured fluorescent substance, for example, a fluorescent dye substance. In this case, the sample solution that has not contributed to the reaction is washed together with the buffer.
[0048]
FIG. 3 shows a specific example of another DNA reaction container 4. The DNA reaction container 4 is made of a plastic material, in which a plurality of sample tanks 401 are arranged, and a DNA microarray 402 is laid in these sample tanks 401. Then, when a sample solution (test sample) is injected into the sample tank 401, a hybridization reaction occurs between the target nucleic acid and the nucleic acid probe that have been fluorescently labeled in advance in the DNA microarray 402, and this reaction causes the DNA microarray Fluorescence is emitted from a captured fluorescent substance, for example, a fluorescent dye. In this case, the sample solution that has not contributed to the reaction is washed together with the buffer.
[0049]
In FIG. 1, only the sample tank 201 of the DNA reaction container 2 and the sample 202 containing a fluorescent substance are shown enlarged on the sample stage 1 for the sake of simplicity.
[0050]
An objective lens 5 is disposed above the sample table 1 as a condenser lens. The objective lens 5 has the optical axis 5a coincident with the center of the sample tank 201, and is located on a perpendicular line from the surface of the sample stage 1.
[0051]
On the other hand, reference numeral 6 denotes a base, on which a cylindrical fixed shaft 7 is provided. The fixed shaft 7 has a flange portion 701 formed at one end, and the flange portion 701 is fixed to the base 6 by a screw 7a.
[0052]
The objective lens 5 is held in a hollow portion of the fixed shaft 7. In this case, the objective lens 5 is movable in the direction of the optical axis 5a, so that the focus can be adjusted.
[0053]
A light source holding member 9 is rotatably supported on the outer peripheral surface of the fixed shaft 7 via a bearing 8. In this case, the light source holding member 9 has a hole 901 formed along the optical axis 5 a, and the peripheral surface of the hole 901 is supported on the outer peripheral surface of the fixed shaft 7 via the bearing 8. The rotation center of the light source holding member 9 coincides with the optical axis 5a of the objective lens 5.
[0054]
Below the bearing 8, a C-ring 8b is arranged. The C ring 8b is for positioning the bearing 8. A bearing spacer 8 a is disposed above the bearing 8. The bearing spacer 8 a is for filling a gap between the fixed shaft 7 and the light source holding member 9.
[0055]
The light source holding member 9 is provided with a plurality of (for example, five) LED light source unit housing holes 902 along the circumferential direction. These LED light source unit storage holes 902 are arranged to be inclined toward the surface of the sample 202 on the sample stage 1. This inclination angle is about 45 ° to 60 ° with respect to the optical axis 5a of the objective lens 5. A screw hole 903 that penetrates the LED light source unit housing hole 902 is provided on the peripheral surface of the light source holding member 9. The screw holes 903 are formed two by two at predetermined intervals in a direction along the LED light source unit housing hole 902 in a horizontal direction orthogonal to the optical axis 5a.
[0056]
The LED light source unit 11 is housed in the LED light source unit housing hole 902. The LED light source unit 11 has a cylindrical LED light source holder 1101. The LED light source 1102 is disposed in the hollow portion of the LED light source holder 1101.
[0057]
Such an LED light source unit 11 is housed in the LED light source unit housing hole 902 with an O-ring 12 interposed. In this case, the O-ring 12 is located between the two screw holes 903.
[0058]
In the LED light source unit housing hole 902, a bandpass filter 1103 is disposed on the optical path of light emitted from the LED light source 1102 of the LED light source unit 11. Here, the bandpass filter 1103 mainly passes near the wavelength range of the light emitted from the LED light source 1102.
[0059]
Further, a spacer 13 is arranged between the holder 1101 of the LED light source unit 11 and the bandpass filter 1103. The spacer 13 positions the LED light source 1102 and the bandpass filter 1103. As a material of the spacer 13, a metal such as aluminum or brass or a plastic is used.
[0060]
In two screw holes 903 penetrating through each LED light source unit housing hole 902, a tilting screw 14 is screwed as a position adjusting means. These tilting screws 14 press two points sandwiching the O-ring 12 on the side of the holder 1101 of the LED light source unit 11. By changing the tilt angle of the light, the light irradiation angle can be adjusted. In this case, the irradiation angle of the light emitted from the LED light source 1102 adjusted by these two tilting screws 14 is about ± 2 to 3 °.
[0061]
The LED light source unit 11 has the same spectral characteristic of the emission wavelength of the LED light source 1102 to which each is applied.
[0062]
A timing pulley 15 is provided integrally around the light source holding member 9. The timing pulley 15 has an uneven portion (not shown) formed on a surface to which a timing belt 18 described later contacts.
[0063]
On the other hand, on the base 6, a motor 16 is provided as a driving means. As the motor 16, a motor capable of controlling the rotation angle such as a servo motor or a pulse motor is used.
[0064]
The motor 16 has a rotating shaft 16 a protruding below the base 6. A timing pulley 17 is provided at the tip of the rotating shaft 16a. The timing pulley 17 also has an uneven portion (not shown) formed on a surface with which a timing belt 18 described later contacts. Further, a timing belt 18 as a rotation transmitting means is stretched between the timing pulleys 17 and 15. The timing belt 18 has irregularities (not shown) fitted to irregularities (not shown) formed on the timing pulleys 17 and 15, respectively. Thus, the light is accurately transmitted to the light source holding member 9 side. In this case, instead of the timing belt 18, a rotation transmitting means such as a gear or a chain can be used. Although a belt can be used, slip may occur. In this case, the rotation of the light source holding member 9 is detected by the sensor 21 and the fluorescence is detected from an integral multiple of the rotation (or reciprocation). Exposure of the means is preferably performed. A fixing member 19 is provided on the base 6. The fixing member 19 has an opposing surface 19 a opposing close to the light source holding member 9. A sliding contact 20 is provided between the facing surface 19a and the light source holding member 9 as power supply means. The sliding contact 20 is, for example, an electrode (not shown) provided on the side facing the power supply electrode (not shown) of the LED light source 1102 of each LED light source unit 11 provided on the light source holding member 9 side (not shown). ) Is in constant contact with the LED light source unit driving circuit 29, which will be described later, so that a driving current can be stably supplied to the LED light source 1102 in a state where the light source holding member 9 is rotated. Other means are also conceivable as the power supply means. For example, an electromagnetic derivative is provided on the light source holding member 9 side, high-frequency power is generated from the fixed portion 19 side, and eddy current is generated on the electromagnetic derivative side. A non-contact type such as sending a message is also conceivable. In short, any device that can supply stable power to the LED light source 1102 without mixing of noise or the like can be used.
[0065]
A sensor 21 is provided on a facing surface 19a of the fixing member 19 as a detecting unit. The sensor 21 detects the rotation of the light source holding member 9, and generates an output every time the light source holding member 9 makes one rotation. The sensor 21 may be of various types, such as a combination of a limit switch and a projection, a combination of a magnet and a Hall element, a combination of a light emitting diode and a light receiving element, and a combination of a photo interrupter and a light shielding plate.
[0066]
On the optical axis 5a on the rear side of the objective lens 5, a filter 22, an imaging lens 23, and a photodetector 24 represented by a CCD camera, which constitute light detection means together with the objective lens 5, are arranged. The filter 22 is for selectively transmitting the fluorescence emitted when the fluorescent substance contained in the sample 202 is excited. The imaging lens 23 forms an image of the fluorescence selected by the filter 22 on the detection surface of the photodetector 24. The photodetector (CCD camera) 24 converts the intensity of the fluorescence collected on the imaging surface into an electric signal and outputs the electric signal to a computer 25 described later. Here, the imaging lens 23 may be a glass lens such as BK7 used for a normal lens as a material, but may be a quartz glass, a plastic lens, or a diffractive optical element, a liquid crystal lens, or the like. An element or a material capable of performing a light condensing action can be used.
[0067]
By the way, the light from each LED light source unit 11 of the light source holding member 9 irradiates the sample 202 on the sample stage 1 as excitation light. In this case, the mounting position of each LED light source unit 11 with respect to the sample 202 needs to illuminate the surface of the sample 202 with uniform brightness by rotating the light source holding member 9. Excitation unevenness is prevented from being caused on the surface of the sample 202 due to the incident angle, and no shadow or uneven excitation is caused on the sample 202 having a three-dimensional portion on a part or the entire surface.
[0068]
Specifically, for example, as shown in FIGS. 4A and 4B, when five LED light source units 11 are used, the irradiation point of the excitation light from each LED light source unit 11 is located on the surface of the sample 202. They are set to be arranged in the radial direction. Here, assuming that the points where the central axes of the light from the LED light source units 11 of # 1 to # 5 penetrate the sample 202 are irradiation points a, b, c, d, and e, as the points approach the periphery of the sample 202, , # 1 to # 5 from the LED light source units 11 to the irradiation points a, b, c, d, and e become longer, and the periphery of the surface of the sample 202 becomes darker than the center. Furthermore, when the surface of the sample 202 is rotated, the peripheral speed of the irradiation point is slower toward the center and faster toward the periphery, so that the irradiation time of the illumination light per unit length is different. Therefore, in order to correct this, the light irradiation points a, b, and c at the peripheral portion of the sample 202 are arranged more densely than the excitation light irradiation points d and e at the central portion, and the light source holding member is provided. When the sample 9 is rotated once in the direction of the arrow in the figure, the entire surface of the sample 202 is made uniform so as to prevent excitation unevenness.
[0069]
In this case, if the entire surface of the sample 202 can be made uniform and no excitation unevenness occurs, the irradiation positions a, b, c, d, and e of the excitation light need to be set so as to be arranged in the radial direction of the surface of the sample 202. However, it is also possible to set randomly on the sample 202 surface. As shown in FIG. 5, when five or more LED light source units 11 are provided, here eight LED light source units 11 # 1 to # 8, irradiation points a, b, c, Irradiation points f, g, and h through which the central axes of the light from the LED light source units # 6 to # 8 penetrate are set on the opposite sides of d and e with respect to the rotation center of the light source holding member 9. In this way, when the light source holding member 9 is rotated once in the direction of the arrow shown in the figure, a stronger excitation light amount can be secured.
[0070]
The main part (fluorescence detection unit) of the fluorescence inspection apparatus thus configured is installed in a light shielding box (not shown) and is shielded from the outside.
[0071]
FIG. 6 shows a block diagram of the entire fluorescence inspection apparatus.
[0072]
In the figure, reference numeral 25 denotes a computer, and the computer 25 is provided with a monitor 27 as display means. An LED light source unit drive circuit 29 is connected to the computer 25 as drive means for driving the fluorescent light detection unit 28 and the LED light source units 11, which are the main components described in FIG. When receiving the control command from the computer 25, the LED light source unit drive circuit 29 determines which of the LED light source units 11 in the fluorescence detection unit 28 is to be driven, and sets and supplies the magnitude of the drive current. I am trying to do it.
[0073]
FIG. 7 is a block diagram of a driving circuit of the LED light source units 11 of # 1 to # 5.
[0074]
In this case, the power supply device 53 is connected to the LED light source unit drive circuit 29, and the computer 25 described in FIG. 6 is connected.
[0075]
The LED light source unit drive circuit 29 receives power from a power supply device 53. The LED light source unit driving circuit 29 is controlled based on a command from the computer 25, supplies a driving current to each of the LED light sources of the LED light source units # 1 to # 5, and generates excitation light. ing.
[0076]
FIG. 8 shows a specific circuit configuration of the power supply device 53 and the LED light source unit drive circuit 29.
[0077]
In this case, the power supply device 53 guides the output from the AC power supply 57 of 100 V to the transformer circuit 58 composed of a transformer to adjust the voltage. The output from the transformer circuit 58 is sent to a bridge rectifier circuit 59 composed of four diodes and full-wave rectified, and then smoothed by a smoothing capacitor 60. The smoothed output is guided to a constant voltage circuit 64 composed of two Darlington-connected power transistors 61 and 62 and an OP amplifier 63, and is output as a constant voltage. In this case, the terminal voltage of the Zener diode 65 is used as a reference voltage, and a variable reference voltage is generated by a variable reference voltage generation circuit 67 including an OP amplifier 66 and a circuit element such as a resistor. By changing the value of the variable reference resistor 68 connected to the OP amplifier 63, the voltage output of the constant voltage circuit 64 is adjusted based on the relationship with the variable reference voltage of the variable reference voltage generation circuit 67, and the LED light source unit is driven. A drive current is supplied to the circuit 29. That is, the drive current to the LED light source unit drive circuit 29 can be simultaneously adjusted by changing the value of the variable reference resistor 68. Here, an FET (field effect transistor: field effect transistor) 70 has a function of preventing an unexpected situation in which an abrupt current flows to the OP amplifier 63 when the current is short-circuited and the OP amplifier 63 is damaged. . The power can be turned on and off by the power switch 69. The power switch 69 is connected to the computer 25, and also controls the power input by the computer 25. The power switch 69 may be manually switched.
[0078]
The LED light source unit drive circuit 29 is connected to such a power supply device 53. In the LED light source unit driving circuit 29, a series circuit of a variable resistor 71a (to 71e) and a switch 72a (to 72e) is separately prepared as a driving circuit of the above-described LED light source units 11 of # 1 to # 5. In this case, the above-mentioned sliding contact 20 is interposed between the LED light source unit drive circuit 29 and the LED light source unit 11 and on the ground side of the LED light source unit 11, respectively.
[0079]
In the LED light source unit drive circuit 29, the drive current supplied to each LED light source of the LED light source units 11 of # 1 to # 5 is adjusted by adjusting the resistance values of the respective variable resistors 71a to 71e. The output light intensity can be controlled. The adjustment of the output light intensity may be performed manually, or may be automatically performed automatically by a computer.
[0080]
The LED light source unit drive circuit 29 controls the on / off of the switches 72a to 72e so that all the LED light sources of the LED light source units # 1 to # 5 can be turned on and off independently. Has become. These on / off controls may be performed automatically by computer control, or may be manually operated.
[0081]
When adjusting the resistance values of the variable resistors 71a to 71e under the control of the computer 25, the intensity of light from the individual LED light sources 1102 of the LED light source units 11 of # 1 to # 5 is detected, and this detection signal is output. Is led to a computer, the light intensity is analyzed by the computer, and the resistance values of the variable resistors 71a to 71e are adjusted. Alternatively, the resistance values of the variable resistors 71a to 71e may be manually adjusted based on the light intensities from the individual LED light sources obtained by this detection. This makes it possible to adjust the inside of the reaction vessel of the DNA microarray so as to irradiate the excitation light almost uniformly. Further, since the variable resistors 71a to 71e can control the magnitude of the drive current flowing through all the LED light sources, it is possible to simultaneously increase or decrease the intensity of the excitation light. Thus, various kinds of samples 202 can be irradiated with excitation light having an appropriate intensity, and fluorescence can be efficiently detected.
[0082]
Next, the operation of the embodiment configured as described above will be described.
[0083]
First, focus adjustment is performed by moving the objective lens 5 held in the hollow portion of the fixed shaft 7 in the direction of the optical axis 5a.
[0084]
Next, the posture of each LED light source unit 11 is adjusted. In this case, the LED light source 1102 of the LED light source unit 11 is turned on without rotating the light source holding member 9, and the excitation light emitted from the LED light source 1102 is irradiated on the surface of the sample 202 on the sample stage 1. In this case, all of the emission spectra of light emitted from the LED light source 1102 are the same.
[0085]
Next, loosen the screw 14 of the LED light source unit housing hole 902, move the LED light source unit 11 in the optical axis direction of the LED light source 1102, adjust the position in the height direction, and reduce the brightness on the surface of the sample 202. adjust. Subsequently, by adjusting the amount of screwing by the tilting screw 14, the inclination angle of the LED light source unit 11 with respect to the surface of the sample 202 is adjusted, and fine adjustment of the light irradiation position on the sample 202 is performed.
[0086]
In this manner, the brightness of the light on the surface of the sample 202 is adjusted, and the light irradiation angle is finely adjusted. As described with reference to FIG. Are set so that the irradiation points a, b, c, d, and e are at predetermined positions on the surface of the sample 202.
[0087]
Next, a control command for performing fluorescence detection is sent from the computer 25 to the LED light source unit drive circuit 29. The LED light source unit drive circuit 29 determines which of the LED light source units 11 in the fluorescence detection unit 28 is to be driven, and at the same time, determines the magnitude of the drive current.
[0088]
Next, the motor 16 is driven by a control command from the computer 25. Then, the timing pulley 17 rotates by the rotation of the motor 16, and the light source holding member 9 rotates together with the timing pulley 15 via the timing belt 18.
[0089]
In this state, a drive current is output from the LED light source unit drive circuit 29. This drive current is supplied to all the LED light sources 1102 of the light source holding member 9 via the sliding contacts 20. When light is emitted from the LED light source 1102, light in a predetermined wavelength range is selected through a band-pass filter 1103, and is radiated toward the sample 202 as excitation light.
[0090]
In this case, as shown in FIG. 4, in the LED light source units # 1 to # 5, the irradiation points a, b, c, d, and e of the excitation light are set at predetermined radial positions on the surface of the sample 202. I have. As a result, the entire surface of the sample 202 is irradiated with the excitation light by one rotation of the light source holding member 9.
[0091]
In this case, the rotation of the light source holding member 9 is set to an integral multiple of one rotation as a minimum unit, so that the entire surface of the sample 202 is uniformly and uniformly excited. The rotation of the light source holding member 9 is detected by the sensor 21 every rotation, and the detection output of the sensor 21 determines the optimal exposure time for the fluorescence detection by the photodetector (CCD camera) 24. That is, the exposure time of the photodetector (CCD camera) 24 is set to an integral multiple of the minimum time required for the light source holding member 9 to make one rotation.
[0092]
By rotating the light source holding member 9 in this manner, the surface of the sample 202 is uniformly and evenly excited by the excitation light of the LED light source units 11 of # 1 to # 5. Light is emitted. The fluorescent light is condensed by the objective lens 5, passes through the filter 22 and the imaging lens 23, and forms an image on the imaging surface of the CCD camera 24. The CCD camera 24 detects the intensity of the fluorescent light emitted from the sample 202 at an exposure time corresponding to the number of rotations of the light source holding member 9, converts this into an electric signal, and outputs it to the computer 25.
[0093]
The computer 25 performs image processing such as contour enhancement, contrast correction, and color correction, performs signal analysis, and the like, and displays the fluorescent image on the monitor 27.
[0094]
Therefore, in this way, a plurality (five in the illustrated example) of the LED light source units 11 having the LED light sources 1102 having the same emission wavelength spectrum are rotatably arranged around the objective lens 5. Since the excitation light from the LED light source unit 11 is applied to the surface of the sample 202 while rotating the LED light source unit 11, efficient excitation light of sufficient intensity to excite the fluorescent substance of the sample 202 can be obtained. Can be.
[0095]
In addition, the irradiation point of the light from each LED light source unit 11 on the surface of the sample 202 is set more densely in the peripheral portion of the surface of the sample 202 than in the central portion, so that the LED light source unit 11 When the periphery of the lens 5 makes one rotation, the entire surface of the sample 202 can be excited uniformly and evenly.
[0096]
Further, the LED light source unit 11 does not need to simultaneously irradiate the entire surface of the sample 202 with light, and the LED light source unit 11 irradiates the entire surface of the sample 202 by rotating the periphery of the objective lens 5. The minimum number of LED light source units 11 can uniformly and efficiently excite the entire surface of the sample 202. In this case, if the number of LED light source units 11 is increased, a stronger excitation light amount can be obtained.
[0097]
Further, since the light from the LED light source 1102 is irradiated from obliquely upward to the surface of the sample 202, the specularly reflected light from the surface of the sample 202 passes through the light receiving optical path on the objective lens 5 side as it is to detect light. The noise light can be reduced without entering the device 24 as noise light.
[0098]
Further, since an LED is used as an excitation light source for exciting a fluorescent substance, it is inexpensive, has a long life, generates little heat, and is safe. Further, power consumption can be reduced. Further, the device configuration can be made compact and highly portable.
[0099]
Further, since the LED light source unit 11 has a configuration using only the band-pass filter 1103 as an optical element other than the LED light source 1102, the configuration is simple, and not only is it easy to assemble and adjust the optical axis, but also easy. It is also useful for reducing the cost.
[0100]
Further, since the LED light source unit 11 is arranged around the objective lens 5 and is separated from the optical path having the objective lens 5 for detecting fluorescence, there is no need to install an extra optical element such as a dichroic mirror. In this regard, a simple device configuration can be achieved. In addition, since there is no optical element such as a dichroic mirror, reflected light and scattered light generated on the surface of these optical elements, and attenuation of fluorescence intensity due to light absorption caused by light passing through these optical elements, etc. Since it does not occur, the loss of the light intensity detected by the photodetector 24 can be minimized. Moreover, since the excitation light path through which the excitation light passes and the light path of the fluorescence detection means are completely separated, adjustment for aligning both optical axes is unnecessary, and the optical axis adjustment of the entire apparatus can be simplified. Therefore, from these, highly reliable fluorescence detection can always be performed.
[0101]
In the above-described embodiment, the rotation of the light source holding member 9 is detected using the sensor 21. However, as the motor 16, a motor such as a servomotor or a pulse motor, which can control the rotation angle, is used. If a device such as a gear that does not cause any slippage is used as the means, the sensor 21 is omitted, the rotation speed of the light source holding member 9 is detected from the control information of the servomotor or the pulse motor, and the optimal exposure time for fluorescence detection is obtained. Can be requested.
[0102]
Also, in the above-described embodiment, the adjustment of the screwing amount of the tilting screw 14 is manually performed. Thus, the adjustment operation may be automatically performed. Further, the LED light source unit 11 may be configured such that at least one of the position adjustment and the inclination angle adjustment in the height direction can be adjusted.
[0103]
Here, the case where the LED light source unit 11 is rotated around the objective lens 5 has been described, but it is not always necessary to rotate around the objective lens 5. For example, when wiring is necessary for supplying power to the LED light source, it is possible to reduce the unevenness of illumination even if it is configured to reciprocate around the objective lens 5, and the present invention can be applied. It is possible. In such a configuration that the LED light source unit 11 reciprocates around the objective lens 5, it is necessary to stop the LED light source unit 11 without fail. If the exposure is performed, the illumination time and the exposure time of the photodetector become long, which is inappropriate. Also, in the case of rotation, if the angular velocity is not set to a predetermined value, the illumination time and the exposure time of the photodetector become inappropriate. Therefore, a configuration in which these shifts and reciprocations are detected by sensors, and illumination and exposure are stopped, or illumination and exposure are performed after reaching a certain angular velocity, and illumination and exposure are stopped when a predetermined rotation or reciprocation is reached is reached. It is also possible.
[0104]
(Modification 1)
As the LED light source unit 11 described above, for example, one having a configuration as shown in FIG. 9 can be used.
[0105]
FIG. 9 shows a schematic configuration of an LED light source unit 31 different from the LED light source unit 11 described above.
[0106]
In this case, the LED light source unit 31 has a cylindrical LED light source holder 3101. The LED light source holder 3101 has an LED light source 3102 disposed in a hollow portion. A bandpass filter 3103, a diffusion plate 3104, and a condenser lens 3105 are arranged on the optical path of light emitted from the LED light source 3102 in the hollow portion of the LED light source holder 3101. Here, the band-pass filter 3103 mainly transmits near the wavelength range of the light emitted from the LED light source 3102. The diffusion plate 3104 is for suppressing the non-uniformity (intensity unevenness) of the intensity of the light from the LED light source 3102 transmitted through the band-pass filter 3103. For example, “frosted glass” or a translucent plastic film is used. ing. The condenser lens 3105 condenses the light diffused by the diffusion plate 3104 and focuses the light on a position determined by the focal length. In this case, the positions of the bandpass filter 3103 and the diffusion plate 3104 may be in the order of the diffusion plate 3104, the bandpass filter 3103, and the condenser lens 3105.
[0107]
In the LED light source unit 31 configured as described above, since the condenser lens 3105 is disposed on the optical path of the light emitted from the LED light source 3102, it is possible to irradiate the surface of the sample 202 with the condenser beam. Thus, for example, if a lens having a large NA of 0.9 or more is used as the condensing lens 3105, light can be condensed in an extremely small region on the surface of the sample 202, for example, in a range of about 0.5 μm in diameter.
[0108]
In addition, since the excitation light from the LED light source 3102 becomes a condensed beam and the area of the irradiation cross section is reduced, the excitation light is applied to a portion other than the surface of the sample 202, such as the side wall of the sample tank 201, and noise light is emitted. As a result, it is possible to prevent the fluorescence from the sample 202 from becoming a factor. Further, if the direction of each LED light source 3102 is adjusted so that the excitation light from the LED light source 3102 is condensed at one point in the surface of the sample 202, the excitation light from the LED light source 3102 can be specified as desired in the surface of the sample 202. Irradiation can be limited to a part. Thus, it is possible to efficiently irradiate the excitation light from the LED light source 3102 to a desired portion on the surface of the sample 202. In addition, since the excitation light from the LED light source 3102 is collected, the fluorescent substance can be efficiently excited. In addition, since light is not diffused, erroneous irradiation of light including portions other than the surface of the sample 202 in the sample tank 201, for example, the side walls and the like can be prevented.
[0109]
(Modification 2)
As the LED light source unit 11 described above, for example, one having a configuration as shown in FIG. 10 can be used.
[0110]
FIG. 10 shows a schematic configuration of an LED light source unit 32 different from the LED light source unit 11 described above.
[0111]
In this case, the LED light source unit 32 has a cylindrical LED light source holder 3201. In the LED light source holder 3201, an LED light source 3202 is arranged in a hollow portion. A bandpass filter 3203, a diffusion plate 3204, and a collimator lens 3205 are arranged on the optical path of light emitted from the LED light source 3202 in the hollow portion of the LED light source holder 3201. Here, the bandpass filter 3203 and the diffusion plate 3204 are the same as those described in FIG. The collimating lens 3205 converts the light diffused by the diffusion plate 3204 into parallel light and emits collimated light. In this case, the positions of the bandpass filter 3203 and the diffusion plate 3204 may be in the order of the diffusion plate 3204, the bandpass filter 3203, and the collimating lens 3205.
[0112]
Since the collimating lens 3205 is arranged on the optical path of the light emitted from the LED light source 3202, the LED light source unit 32 configured as described above can irradiate the surface of the sample 202 with uniform collimated light. This makes it easy to specify the irradiation position in the plane of the sample 202, and it is possible to easily program a program for automatically adjusting the irradiation position by computer control, and to easily predict the irradiation surface position and its illuminance. Is beneficial. Furthermore, even when the position to irradiate the excitation light is manually set, the operation of the excitation light can be accurately performed at a desired irradiation position on the surface of the sample 202. In addition, since light is not diffused, erroneous irradiation of light including portions other than the surface of the sample 202 in the sample tank 201, for example, the side walls and the like can be prevented.
[0113]
(Modification 3)
As the LED light source unit 11 described above, for example, one having a configuration as shown in FIG. 11 can be used.
[0114]
FIG. 11 shows a schematic configuration of an LED light source unit 33 different from the LED light source unit 11 described above.
[0115]
In this case, the LED light source unit 33 has a cylindrical LED light source holder 3301. In the LED light source holder 3301, an LED light source 3302 is arranged in a hollow portion. A bandpass filter 3303 and a diffusion plate 3304 are arranged on the optical path of light emitted from the LED light source 3302 in the hollow portion of the LED light source holder 3301. Here, the bandpass filter 3303 and the diffusion plate 3304 are the same as those described in FIG. In this case, the positions of the bandpass filter 3303 and the diffusion plate 3304 may be in the order of the diffusion plate 3304 and the bandpass filter 3303.
[0116]
In the LED light source unit 33 configured as described above, since the diffusion plate 3304 is arranged on the optical path of the light emitted from the LED light source 3302, it is possible to irradiate the diffuse light without unevenness on the surface of the sample 202. As a result, it is possible to irradiate substantially uniform excitation light evenly over the entire surface of the sample 202, and it is possible to receive stable and excellent fluorescence with excellent reproducibility.
[0117]
In addition, these LED light source units 11, 31, 32, and 33 can be properly used according to the size and structure of the sample 202 and the sample tank 201. That is, since the LED light source units 11, 31, 32, and 33 are integrally configured by combining the LED light sources with optical elements such as a band-pass filter, a lens, and a diffusion plate, excitation is performed for each LED light source unit. Light can be condensed, collimated, or diffused, and the beam pattern of the excitation light on the surface of the sample 202 can be controlled in various ways. It can also be used for a sample having irregularities. Further, for the LED light source unit 31 (32), either one of the bandpass filter 3103 (3203) and the diffusion plate 3104 (3204) may be used, or both may not be used depending on the application. Further, the band-pass filters 1103, 3103, 3203, and 3303 of the LED light source units 11, 31, 32, and 33 may be high-pass filters or low-pass filters depending on the spectral region of the emission wavelength of the fluorescent dye used as the sample. It is. Furthermore, the condenser lens 3105 and the collimator lens 3205 may be glass lenses such as BK7 used as a material as a normal lens, but may be quartz glass, a plastic lens, or a diffractive optical element, a liquid crystal lens, or the like. An element or a material capable of condensing visible light can be used. In the above-described embodiment, the case where the plurality of LED light source units 11 are arranged around the objective lens 5 has been described. However, when the sample 202 is extremely small, it is arranged around the objective lens 5 in FIG. It is also possible to use only one LED light source unit 11. Even in this case, the same effect as described above can be expected.
[0118]
(Second embodiment)
Next, a second embodiment of the present invention will be described.
[0119]
By the way, in order to illuminate the sample surface with light of uniform brightness without unevenness, it is necessary to confirm this state in advance.
[0120]
FIG. 12 shows a schematic configuration of the second embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0121]
In this case, instead of the sample 202, a photodetector 34 is disposed as a light intensity detecting unit on the sample stage 1 to which light from the plurality of LED light source units 11 is irradiated. Here, as the photodetector 34, a solid-state imaging device (CCD camera or CMOS sensor), an imaging tube, or the like is used.
[0122]
The light detectors 34 individually detect the intensity of light from each LED light source unit 11. The detection output of the photodetector 34 is taken into the computer 25 described with reference to FIG.
[0123]
The computer 25 analyzes the variation of the light intensity from the output of the photodetector 34 according to the intensity of the light from each LED light source unit 11 and sends a signal to the LED light source unit driving circuit 29 shown in FIG. The current supplied to the LED light source 1102 of the light source unit 11 is individually controlled.
[0124]
Accordingly, the magnitude of the current supplied to each LED light source 1102 can be controlled based on the light intensity obtained near the sample, so that the light from each LED light source 1102 can be made to have the same brightness, and the surface of the sample 202 can be controlled. Can be illuminated with uniform brightness. Therefore, illumination unevenness on the surface of the sample 202 can be suppressed with higher accuracy, the fluorescent substance of the sample 202 can be excited almost uniformly, and highly reliable fluorescence detection can be performed.
[0125]
In the above description, the computer 25 was used, but the LED light source unit drive circuit 29 was manually adjusted based on the intensity of light from the LED light source 1102 of each LED light source unit 11 obtained by the photodetector 34, and the LED light source was adjusted. The light from 1102 may be adjusted to the same brightness, then the photodetector 34 may be removed, and the sample tank 201 may be re-installed at this position.
[0126]
On the other hand, an image pickup means (for example, a CCD camera or a CMOS sensor (both not shown)) is used in place of the photodetector 24, and the sample tank 201 is directly mounted on the sample table 1, and each LED light source unit 11 The sample tank 201 is individually irradiated with light from the LED light source 1102, and the fluorescence from the sample tank 201 passes through the objective lens 5, the filter 22, and the imaging lens 23, respectively, is imaged by the imaging means, and guided to the computer 25. The brightness of each pixel of the fluorescent image on the monitor 27 is analyzed by the computer 25 to determine the variation of the light intensity from each LED light source 1102, and the LED is determined based on the result. By adjusting the light source unit drive circuit 29, the current supplied to each LED light source 1102 can be individually controlled.
[0127]
Even in this case, the light from each LED light source 1102 can be adjusted to the same brightness, so that illumination unevenness on the surface of the sample 202 can be suppressed, and the fluorescent substance in the sample tank 201 can be made substantially uniform. Can be excited.
[0128]
(Third embodiment)
Next, a third embodiment of the present invention will be described.
[0129]
In the third embodiment, two or more LED light sources having different peak emission wavelengths are used to excite a plurality of types of fluorescent dyes so that a fluorescent signal can be detected.
[0130]
FIG. 13 shows a schematic configuration of the third embodiment, and the same parts as those of FIG. 1 are denoted by the same reference numerals.
[0131]
In this case, first to third LED light source units 41, 42, and 43 having three types of LED light sources having different peak emission wavelengths are mounted on the light source holding member 9. Generally, the emission spectrum of an LED light source has a mountain-shaped structure and has one peak wavelength. Here, an LED light source having a peak emission wavelength of 490 nm is used for the first LED light source unit 41, an LED light source having a peak emission wavelength of 520 nm is used for the second LED light source unit 42, and a third LED light source unit is used. As the LED light source unit 43, an LED light source having a peak emission wavelength of 630 nm is used.
[0132]
The first to third LED light source units 41, 42, 43 are arranged at four symmetrical positions along the periphery of the light source holding member 9 with the objective lens 5 interposed therebetween. In other words, these first to third LED light source units 41, 42, and 43 have LED light sources having different spectral characteristics of emission wavelengths, respectively, and the first to third LED light source units 41, 42 having different spectral characteristics. 42 and 43 are arranged at regular intervals along the periphery of the light source holding member 9 in order. Here, the first LED light source unit 41, the second LED light source unit 42, and the third LED light source unit 43 are repeatedly arranged in this order. The first to third LED light source units 41, 42, and 43 are arranged at positions facing each other with the objective lens 5 therebetween so that those having the same spectral characteristics come.
[0133]
A band-pass filter (not shown) installed in front of each LED light source of the first to third LED light source units 41, 42, 43 transmits the vicinity of the peak wavelength of light emission of each LED light source best. It has characteristics. That is, the band-pass filter of the first LED light source unit 41 has a wavelength of the best transmission set at around 490 nm, and the band-pass filter of the second LED light source unit 42 has a wavelength of the best transmission set at around 520 nm. The band-pass filter of the third LED light source unit 43 has a wavelength at which the light is transmitted best is set to around 630 nm.
[0134]
FIG. 14 shows a schematic configuration of a photodetection optical system of the device configured as described above. In this example, the LED light source units 41, 42, and 43 having the above-mentioned three different wavelengths at their peak emission wavelengths use a sample. The case of irradiating the excitation light to is shown.
[0135]
In this case, two dichroic mirrors 45 and 46 are arranged on the optical axis above the objective lens 5.
[0136]
The dichroic mirror 45 is installed so that the reflected light travels in a direction of approximately 45 ° with respect to the optical axis of the objective lens 5, and has a wavelength slightly longer than the peak emission wavelength of the fluorescent dye FITC of 520 nm, for example, 550 nm or less. It has a transmission reflection spectrum characteristic that reflects light of a wavelength and transmits light of a longer wavelength. The dichroic mirror 46 is installed so that the reflected light travels in a direction substantially 45 ° with respect to the optical axis of the objective lens 5, and has a wavelength slightly longer than the peak emission wavelength 565 nm of the fluorescent dye Cy3, for example, 620 nm. It has a transmission reflection spectrum characteristic that reflects light of the following wavelengths and transmits light of a longer wavelength.
[0137]
Further, a CCD camera 48 is disposed on a reflection optical path of the dichroic mirror 45 via a condenser lens 47. In addition, a CCD camera 50 is arranged on a reflection optical path of the dichroic mirror 46 via a condenser lens 49, and a CCD camera 52 is arranged on a transmission optical path via a condenser lens 51.
[0138]
The outputs of the CCD cameras 48, 50, 52 are sent to the computer 25.
[0139]
In such a configuration, three types of target DNAs are set for one sample, and different fluorescent dyes FITC (Fluorescein-isothiocyanate), Cy3, and Cy5 are respectively labeled with these. Next, a sample solution containing DNA labeled with these fluorescent dyes is dropped into a sample tank, DNA hybridization is performed, and the labeling substance that has not contributed to the reaction is removed with a buffer solution (PBS (phosphate buffer solution). ), A mixed solution of EDTA (disodium ethylenediaminetetraacetate) and NaCl: PH 7.4).
[0140]
The sample thus obtained is set on the sample table 1. Then, while rotating the first to third LED light source units 41, 42, 43 by the light source holding member 9 shown in FIG. 13, the excitation light from the first to third LED light source units 41, 42, 43 is supplied to the sample 202. Irradiation. At this time, the light from the LED light source having the three different wavelengths as the peak emission wavelengths is all simultaneously irradiated on the surface of the sample 202.
[0141]
Then, fluorescence is emitted from the fluorescent dye attached to the DNA attached to the reaction tank of the DNA microarray by the DNA hybridization, and the light reaches the dichroic mirror 45 through the objective lens 5 shown in FIG. . In this case, the fluorescence by the fluorescent dye FITC is reflected by the dichroic mirror 45, passes through the lens 47, enters the CCD camera 48, is guided to the computer 25, and is obtained as a green fluorescent image by FITC. Fluorescent light from the fluorescent dye Cy3 passes through the dichroic mirror 45, is reflected by the dichroic mirror 46, passes through the lens 49, enters the CCD camera 50, is guided to the computer 25, and is converted into an orange fluorescent image by Cy3. Is obtained as Further, the fluorescent light from the fluorescent dye Cy5 passes through the dichroic mirror 45 and the dichroic mirror 46, passes through the lens 51, enters the CCD camera 52, and is guided to the computer 25 to obtain a red fluorescent image by Cy5. Can be In the computer 25, the fluorescent images from the three types of fluorescent dyes are synthesized by the CCD cameras 48, 50 and 52 and output to the monitor 27, or the fluorescent images from the three types of fluorescent dyes are output. These are separately displayed on the monitor 27.
[0142]
FIG. 15 is a block diagram of a drive circuit of the first to third LED light source units 41, 42, and 43.
[0143]
In this case, an LED light source unit drive circuit 54, an LED light source unit drive circuit 55, and an LED light source unit drive circuit 56 are provided as drive circuits for the first to third LED light source units 41, 42, and 43, respectively. An LED light source unit drive circuit 54 is connected to the first LED light source unit 41, an LED light source unit drive circuit 55 is connected to the second LED light source unit 42, and a third LED light source unit 43 is The LED light source unit drive circuit 56 is connected. A common power supply 53 is connected to the LED light source unit driving circuit 54, the LED light source unit driving circuit 55, and the LED light source unit driving circuit 56, and the computer 25 described with reference to FIG.
[0144]
The LED light source unit driving circuit 54, the LED light source unit driving circuit 55, and the LED light source unit driving circuit 56 are supplied with power from a common power supply device 53. The LED light source unit driving circuit 54, the LED light source unit driving circuit 55, and the LED light source unit driving circuit 56 are controlled based on a command from the computer 25, and are controlled by the first LED light source unit 41 and the second LED light source unit 42. A driving current is supplied to each LED light source of the third LED light source unit 43 to generate excitation light of three types of fluorescent dyes.
[0145]
In this case, the three kinds of fluorescent signals obtained by these excitation lights reach the CCD cameras 48, 50 and 52 by dichroic mirrors 45 and 46, respectively, as shown in FIG. , 50, and 52 are guided to a computer 25, where image analysis is performed, and a composite image using three types of fluorescent dyes is generated and output.
[0146]
FIG. 16 shows a specific circuit configuration of the power supply device 53 and the LED light source unit drive circuits 54, 55, 56.
[0147]
In this case, with respect to the power supply device 53, the same portions as those in FIG.
[0148]
LED light source unit drive circuits 54, 55, 56 are connected in parallel to such a power supply device 53. In the LED light source unit drive circuit 54, a series circuit of a variable resistor 71 and a switch 72 is separately prepared as a drive circuit of the four first LED light source units 41 described in FIG. Similarly, in the LED light source unit driving circuit 55, a series circuit of a variable resistor 71 and a switch 72 is separately prepared as a driving circuit for the four second LED light source units 42, and the LED light source unit driving circuit 56 is also provided. A series circuit of a variable resistor 71 and a switch 72 is separately prepared as a drive circuit for the four third LED light source units 43.
[0149]
In these LED light source unit driving circuits 54, 55, and 56, the resistance values of the respective variable resistors 71, 73, and 75 are adjusted so that the LED light sources of the first to third LED light source units 41, 42, and 43 are adjusted. The supplied driving current can be adjusted to control the output light intensity. The adjustment of the output light intensity may be performed manually, or may be automatically performed automatically by a computer. Thereby, the drive current of each LED light source of the first to third LED light source units 41, 42, 43 can be individually controlled.
[0150]
The LED light source unit driving circuits 54, 55, and 56 control the on / off of the switches 72, 74, and 76 so that the LED light sources of the first to third LED light source units 41, 42, and 43 are all turned on. It can be turned on and off independently. These on / off controls may be performed automatically by computer control, or may be manually operated.
[0151]
When adjusting the resistance values of the variable resistors 71, 73, and 75 by computer control, the individual LED light sources of the first to third LED light source units 41, 42, and 43 are used as described with reference to FIG. The detection signal is guided to the computer 25, and the computer 25 analyzes the light intensity to adjust the resistance values of the variable resistors 71, 73, and 75. Alternatively, the resistance values of the variable resistors 71, 73, and 75 may be manually adjusted based on the light intensities from the individual LED light sources obtained by this detection. This makes it possible to adjust the light from the LED light source to irradiate the inside of the reaction vessel of the DNA microarray almost uniformly. Further, it is possible to partially adjust the light intensity, for example, by concentrating the excitation light at a desired position in the sample tank 201. Further, the variable resistors 71, 73, and 75 can control the magnitude of the drive current flowing through all the LED light sources, so that the intensity of the excitation light can be increased or decreased all at once. As a result, it is possible to irradiate the excitation light of an appropriate intensity to each sample, and it is possible to efficiently detect the fluorescence.
[0152]
Therefore, in this way, three types of fluorescent dyes can be excited simultaneously using the first to third LED light source units 41, 42, 43 having three types of LED light sources having different peak emission wavelengths. It is possible to acquire fluorescent images from these three types of fluorescent dyes, synthesize them, and display them individually.
[0153]
Also, the individual LED light sources constituting the first to third LED light source units 41, 42, 43 can be easily turned on and off according to the fluorescent dye to be excited. Since the magnitude of the drive current can also be individually adjusted by the operation, it is possible to irradiate each fluorescent dye with excitation light of an appropriate intensity, and it is possible to detect fluorescence efficiently.
[0154]
In the above-described third embodiment, three types of fluorescent dyes are used. However, the present invention is not limited to this, and two types may be used. Can be. That is, a dichroic mirror, a lens, and a CCD camera may be added for each type of fluorescent dye. Also, the fluorescent dye is not limited to FITC, Cy3 and Cy5 shown in the embodiment, but may be Rhodamine green (Rhodamine G), Texas red (Texas Red), RITC (Rhodamine B-isothiocia) or the like. .
[0155]
The number of LED light source units is not particularly limited, and a plurality of LED light source units can be individually controlled by a plurality of drive circuits. The output wavelengths of a plurality of LED light source units may be the same, or the present invention can be applied even if they are different. In addition, it is also possible to irradiate the sample with one kind of light from LED light sources having different peak emission wavelengths, and to repeat the light emission by the number of different wavelengths.
[0156]
(Fourth embodiment)
Next, a fourth embodiment of the present invention will be described.
[0157]
In the fourth embodiment, a reflecting member is arranged on the optical path of light emitted from the LED light source unit, and the light in the optical path is reflected, so that the optical axis angle of the excitation light is more reduced to the optical axis angle of the objective lens. I try to get closer.
[0158]
FIG. 17 shows a schematic configuration of the fourth embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0159]
In this case, around the objective lens 5, a plurality of LED light source units 81 and a reflection plate 82 as a reflection member corresponding to the LED light source units 81 are respectively held by the light source holding members 9 described in FIG. Then, the objective lens 5 is rotatable around the optical axis 5a.
[0160]
The LED light source unit 81 has a cylindrical LED light source holder 8101. The LED light source holder 8101 has an LED light source 8102 disposed in a hollow portion. A bandpass filter 8103, a diffusion plate 8104, and a condenser lens 8105 are arranged on the optical path of light emitted from the LED light source 8102 in the hollow portion of the LED light source holder 8101. Here, the band-pass filter 8103 mainly passes near the wavelength range of the light emitted from the LED light source 8102.
[0161]
On the optical path of light emitted from these LED light source units 81, reflection plates 82 are separately arranged. The reflection plate 82 reflects light from the LED light source unit 81 and irradiates the sample 202 in the sample tank 201 on the sample stage 1 as excitation light, and simultaneously excites the optical axis of the objective lens 5. The angle θ formed by the optical axis of the light is reduced so that the optical axis angle of the excitation light approaches the optical axis angle of the objective lens.
[0162]
Here, the respective mounting positions of the reflection plates 82 are such that the irradiation points on the sample 202 of the respective lights after reflecting the light from the LED light source unit 81 are the same as described in FIGS. 4A and 4B. It is set so as to be arranged in the radial direction of the sample 202 surface.
[0163]
Others are the same as FIG.
[0164]
Therefore, even in this case, the same effect as in the first embodiment can be expected. Further, since the reflection plate 82 is arranged on the optical path of the light emitted from the LED light source unit 81 and reflects the light in the optical path, the optical axis angle of the excitation light can be made closer to the optical axis angle of the objective lens 5. . This makes it possible to irradiate the excitation light from a position immediately above the surface of the sample 202 (a limit that does not enter the observation field of view of the objective lens 5). The difference between the shortest distance “a” and the longest distance “b” of the optical path can be made small. As a result, the intensity of the excitation light is inversely proportional to the distance of the optical path, so that the intensity difference of the excitation light on the surface of the sample 202 can be reduced, and the surface of the sample 202 can be efficiently and uniformly excited. .
[0165]
Also in the fourth embodiment, a control method for illuminating the sample with light of uniform brightness without unevenness as described in the modification of the second embodiment and the third embodiment is described. Can be adopted.
[0166]
(Modification 1)
By the way, in the fourth embodiment, the reflection plate 82 is arranged as a reflection member on the optical path of the light emitted from the LED light source unit 81 so as to reflect the light in the optical path. Since the degree of freedom in the arrangement of the LED light source units 81 is increased, a large number of LED light source units 81 can be arranged around the objective lens 5.
[0167]
FIG. 18 shows a schematic configuration of Modification Example 1, and the same parts as those in FIG. 17 are denoted by the same reference numerals.
[0168]
In this case, a plurality of LED light source units 81 (two layers in the illustrated example) are arranged around the objective lens 5 as semiconductor light source means. On the optical path of light emitted from these LED light source units 81, reflecting plates 82 and 82 '(corresponding to two layers in the illustrated example) are separately arranged as reflecting members corresponding to the LED light source units 81 of each layer. I have. The LED light source unit 81 and the reflection plates 82 and 82 ′ of each layer are held by the light source holding member 9 described with reference to FIG. 1 and are rotatable about the optical axis 5 a of the objective lens 5.
[0169]
In this case, as for the respective mounting positions of the reflection plates 82 and 82 ', the irradiation points on the sample 202 of the respective lights after reflecting the light from the LED light source unit 81 are shown in FIGS. In the same manner as described above, it is set so as to be arranged in the radial direction of the sample 202 surface.
[0170]
Others are the same as FIG.
[0171]
Even in this case, the same effect as in the first embodiment can be expected. Further, since the light from the light source unit 81 is reflected by using the reflection plates 82 and 82 ', the degree of freedom of arrangement of the LED light source units 81 can be increased. By increasing the degree of freedom in the arrangement of these LED light source units 81, it is possible to use more LED light source units 81 than in the case of the fourth embodiment. Thus, the surface of the sample 202 can be evenly irradiated with the stronger excitation light.
[0172]
In addition, since the number of LED light source units 81 can be greatly increased, the LED light source units 81 are divided into a plurality of sets, and the LED light sources having different emission wavelength spectral characteristics are used for each set. As described in the third embodiment, it is possible to excite a plurality of types of fluorescent dyes and detect fluorescent signals.
[0173]
(Modification 2)
On the other hand, in the fourth embodiment, the reflecting plate 82 is arranged as a reflecting member on the optical path of the light emitted from the LED light source unit 81 to reflect the light in the optical path, but a dichroic mirror is used as the reflecting member. Can be.
[0174]
FIG. 19 shows a schematic configuration of Modification Example 2, and the same parts as those in FIG. 17 are denoted by the same reference numerals.
[0175]
In this case, a plurality of LED light source units 81 as semiconductor light source means and a dichroic mirror 83 as a reflecting member corresponding to these LED light source units 81 are provided around the objective lens 5 as the light sources described in FIG. It is held by a holding member 9 and is rotatable about the optical axis 5a of the objective lens 5.
[0176]
On the optical path of the light emitted from the LED light source 8102 in the hollow portion of the LED light source holder 8101, a diffusion plate 8104 and a condenser lens 8105 are arranged, and the above-described bandpass filter 8103 is omitted.
[0177]
On the optical path of light emitted from the LED light source unit 81, dichroic mirrors 83 are separately arranged as reflection members. The dichroic mirror 83 has a characteristic of mainly reflecting near the wavelength range of light emitted from the LED light source 8102 of the LED light source unit 81. The dichroic mirror 83 reflects the light from the LED light source unit 81 and irradiates the sample 202 in the sample tank 201 on the sample stage 1 as excitation light. Also in this case, the angle formed by the optical axis of the excitation light with respect to the optical axis of the objective lens 5 is reduced so that the optical axis angle of the excitation light approaches the optical axis angle of the objective lens.
[0178]
Also in this case, the mounting position of the dichroic mirror 83 is such that the irradiation point on the sample 202 of each light after reflecting the light from the LED light source unit 81 is the same as described in FIGS. 4A and 4B. It is set so as to be arranged in the radial direction of the sample 202 surface.
[0179]
Others are the same as FIG.
[0180]
Even if the dichroic mirror 83 is used as the reflecting member instead of the reflecting plate, the same effect as in the fourth embodiment can be expected. Further, by using a dichroic mirror 83 having a characteristic of mainly reflecting near the wavelength range of light emitted from the LED light source 8102 of the LED light source unit 81, the band-pass filter built in the LED light source unit 81 is used. Can be omitted. Accordingly, there is an effect that the cost of the LED light source unit 81 can be reduced and the size can be reduced.
[0181]
(Fifth embodiment)
Next, a fifth embodiment of the present invention will be described.
[0182]
In the fifth embodiment, the light guide member is arranged on the optical path of the light emitted from the LED light source unit, and the optical path is bent so that the optical axis angle of the excitation light is closer to the optical axis angle of the objective lens. I have to.
[0183]
FIG. 20 shows a schematic configuration of the fifth embodiment, and the same parts as those of FIG. 1 are denoted by the same reference numerals.
[0184]
In this case, a plurality of LED light source units 81 as semiconductor light source means and an optical fiber 84 as a light guide member corresponding to these LED light source units 81 are described in FIG. It is held by a light source holding member 9 and is rotatable about the optical axis 5a of the objective lens 5.
[0185]
The LED light source unit 81 has a cylindrical LED light source holder 8101. The LED light source holder 8101 has an LED light source 8102 disposed in a hollow portion. A bandpass filter 8103, a diffusion plate 8104, and a condenser lens 8105 are arranged on the optical path of light emitted from the LED light source 8102 in the hollow portion of the LED light source holder 8101. Here, the band-pass filter 8103 mainly passes near the wavelength range of the light emitted from the LED light source 8102.
[0186]
On the optical path of the light emitted from these LED light source units 81, the light incident ends 84a of the optical fibers 84 are separately arranged. The optical fiber 84 allows the optical path of the light from the LED light source unit 81 to be freely bent, and converts the light from the light emitting end 84b into the excitation light to the sample 202 in the sample tank 201 on the sample stage 1. At the same time, the angle between the optical axis of the excitation light and the optical axis of the objective lens 5 is reduced so that the optical axis angle of the excitation light approaches the optical axis angle of the objective lens.
[0187]
The mounting position of the light emitting end 84b of the optical fiber 84 is such that the irradiation point of the light emitted from the light emitting end 84b on the sample 202 is on the surface of the sample 202 in the same manner as described with reference to FIGS. They are set to be arranged in the radial direction.
[0188]
Others are the same as FIG.
[0189]
Therefore, even in this case, the same effect as in the first embodiment can be expected. Further, since the optical fiber 84 is arranged on the optical path of the light emitted from the LED light source unit 81 and the optical path is bent, the optical axis angle of the excitation light can be made closer to the optical axis angle of the objective lens. Thus, the intensity difference of the excitation light on the surface of the sample 202 can be reduced in the same manner as described in the fourth embodiment, and the surface of the sample 202 can be efficiently and uniformly excited.
[0190]
In the above description, an example in which the optical fiber 84 is used as the light guide member has been described. However, for example, a member formed by processing a glass rod or an acrylic material may be used.
[0191]
It should be noted that also in the fifth embodiment, a control method for illuminating the sample with light of uniform brightness without unevenness as described in the modification of the second embodiment and the third embodiment is described. Can be applied.
[0192]
(Modification)
By the way, in the fifth embodiment, the optical fiber 84 is arranged as a light guide member on the optical path of the light emitted from the LED light source unit 81 to bend the optical path. Since the degree of freedom in the arrangement of the light source units 81 increases, a large number of LED light source units 81 can be arranged around the objective lens 5.
[0193]
FIG. 21 shows a schematic configuration of a modification, and the same parts as those in FIG. 20 are denoted by the same reference numerals.
[0194]
In this case, a plurality of LED light source units 81 as semiconductor light source means are arranged in a multiplex (double in the illustrated example) around the objective lens 5, and optical fibers as light guide members corresponding to these LED light source units 81 are provided. Along with the light 84, it is held by the light source holding member 9 described in FIG. 1 and is rotatable about the optical axis 5 a of the objective lens 5.
[0195]
Also in this case, the mounting position of each of the light emitting ends 84b of the optical fibers 84 is such that the irradiation point of the light emitted from the light emitting end 84b on the sample 202 is described with reference to FIGS. 4 (a) and 4 (b). Similarly, it is set so as to be arranged in the radial direction of the sample 202 surface.
[0196]
Others are the same as FIG.
[0197]
In this way, since the optical path of the light from the light source unit 81 is bent by using the optical fiber 84, the degree of freedom of the position where each LED light source unit 81 is arranged can be increased. Then, by increasing the degree of freedom of the position where these LED light source units 81 are arranged, it becomes possible to use more LED light source units 81 than in the case of the fifth embodiment. This makes it possible to easily obtain the amount of light required to irradiate the surface of the sample 202 and to illuminate the surface of the sample 202 without unevenness in the amount of light.
[0198]
In addition, since the number of LED light source units 81 can be greatly increased, the LED light source units 81 are divided into a plurality of sets, and the LED light sources having different emission wavelength spectral characteristics are used for each set. As described in the third embodiment, it is possible to excite a plurality of types of fluorescent dyes and detect fluorescent signals.
[0199]
In this modification, the example in which the optical fiber 84 is used as the light guide member has been described. However, for example, a material obtained by molding a glass rod or an acrylic material can be used.
[0200]
(Sixth embodiment)
Next, a sixth embodiment of the present invention will be described.
[0201]
In the above-described embodiment, the sample stage 1 is fixed, and the plurality of LED light source units 11 are rotatably provided with respect to the sample 202 on the sample stage 1. However, in the sixth embodiment, The plurality of LED light source units 11 are fixed, and the sample stage 1 is rotatable.
[0202]
FIG. 22 shows a schematic configuration of the sixth embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0203]
In this case, the cylindrical fixed shaft 7 holds the objective lens 5 in the hollow portion, and the light source holding member 9 is integrally provided on the peripheral surface portion. The light source holding member 9 holds a plurality of LED light source units 11.
[0204]
On the other hand, a rotating shaft 91 is connected to the sample stage 1 arranged on the optical axis 5a of the objective lens 5. The rotating shaft 91 is rotatably supported by a base 93 via a bearing 92. Further, a timing pulley 94 is provided on the rotating shaft 91.
[0205]
The base 93 is provided with a motor 95 as driving means. As the motor 95, a motor capable of controlling the rotation angle such as a servomotor or a pulse motor is used.
[0206]
In the motor 95, a timing pulley 96 is provided at a tip of a rotating shaft 95a. Further, a timing belt 97 as a rotation transmitting means is stretched between the timing pulleys 94 and 96. The timing belt 97 has irregularities (not shown) fitted to irregularities (not shown) formed on the timing pulleys 94 and 96, respectively, and accurately rotates the motor 95 to the sample stage 1 via the timing pulley 94. I'm going to tell you. In this case, instead of the timing belt 97, a rotation transmitting unit such as a gear or a chain can be used.
[0207]
Also in this case, while irradiating the sample 202 with excitation light from the LED light source unit 11 on the sample 202 under the same conditions as described in the first embodiment, the entire surface of the sample 202 is Excitation can be performed uniformly without causing unevenness, and the same effect as in the first embodiment can be expected. Further, in this case, the sliding contact 20 as the power supply means can be omitted, and the LED light source unit 11 can be directly connected to the LED light source unit drive circuit 29 using a cable. Can be performed.
[0208]
In the above-described embodiment, the LED light source unit 11 or the sample stage 1 is rotated. However, the LED light source unit 11 or the sample stage 1 is rotated at a predetermined rotation angle, for example, 360 °. It may be made to reciprocate at a corner. In this way, especially when rotating the LED light source unit 11 side, the sliding contact 20 can be omitted, and the LED light source unit 11 can be directly connected to the LED light source unit drive circuit 29 by a cable, so that noise and the like are more mixed. Small and stable power supply can be performed. Of course, in this case, even if the LED light source unit 11 side or the sample stage 1 side is reciprocated in the linear direction, the same effect as described above can be expected.
[0209]
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. For example, the sample applied in each of the above-described embodiments is not limited to an example of a DNA microarray, but can be applied to all reaction vessels called so-called DNA microarrays. In addition, the present invention is widely applicable not only to DNA but also to inspection and measurement using various other biological materials.
[0210]
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.
[0211]
The embodiments described above include the following inventions.
[0212]
(1) A plurality of the semiconductor light source means are arranged along the periphery of the condenser lens, and the sample stage is provided rotatably around an optical axis of the condenser lens.
[0213]
(2) The plurality of semiconductor light source means are arranged around the condenser lens, and are further arranged on the optical path of light emitted from the semiconductor light source means, and reflect the light on the optical path to cause the sample to emit excitation light. It is characterized by having a reflecting member for irradiating as.
[0214]
(3) A plurality of the semiconductor light source means are arranged around the condenser lens, further have a characteristic of reflecting near the wavelength range of light emitted from the semiconductor light source means, and are emitted from the semiconductor light source means. A dichroic mirror is provided on the optical path of the light, and reflects the light in the optical path to irradiate the sample as excitation light.
[0215]
(4) The plurality of semiconductor light source means are arranged around the condenser lens, and further arranged in the optical path of light emitted from the semiconductor light source means, and the optical path is bent so that the light in the optical path is transmitted to the sample. It is characterized by having a light guide member for irradiating as excitation light.
[0216]
【The invention's effect】
As described above, according to the present invention, it is an object of the present invention to provide a fluorescence detection device that can efficiently irradiate a sufficient excitation light without unevenness and perform highly reliable fluorescence detection.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a fluorescence inspection apparatus based on a microscope structure according to a first embodiment of the present invention.
FIG. 2 is a diagram showing a schematic configuration of an example of a DNA reaction container used in the first embodiment.
FIG. 3 is a diagram showing a schematic configuration of another example of the DNA reaction container used in the first embodiment.
FIG. 4 is a diagram for explaining irradiation of excitation light onto a sample according to the first embodiment.
FIG. 5 is a view for explaining another example of irradiation of the sample with excitation light according to the first embodiment;
FIG. 6 is a block diagram showing the entire fluorescence inspection apparatus according to the first embodiment.
FIG. 7 is a block diagram showing an LED light source unit drive circuit used in the first embodiment.
FIG. 8 is a diagram showing a schematic configuration of an LED light source driving circuit used in the first embodiment.
FIG. 9 is a diagram showing a schematic configuration of an LED light source unit as a first modification of the first embodiment.
FIG. 10 is a diagram showing a schematic configuration of an LED light source unit as a second modification of the first embodiment.
FIG. 11 is a diagram showing a schematic configuration of an LED light source unit as a third modification of the first embodiment.
FIG. 12 is a diagram illustrating a schematic configuration of a main part of a fluorescence inspection apparatus according to a second embodiment of the present invention.
FIG. 13 is a diagram illustrating a schematic configuration of a fluorescence inspection apparatus according to a third embodiment of the present invention.
FIG. 14 is a diagram showing a schematic configuration of a measurement optical system used in a third embodiment.
FIG. 15 is a block diagram showing an LED light source unit drive circuit used in the third embodiment.
FIG. 16 is a diagram showing a schematic configuration of an LED light source driving circuit used in the third embodiment.
FIG. 17 is a diagram illustrating a schematic configuration of a fluorescence inspection apparatus according to a fourth embodiment of the present invention.
FIG. 18 is a diagram illustrating a schematic configuration of a first modification of the fourth embodiment;
FIG. 19 is a diagram illustrating a schematic configuration of a second modification of the fourth embodiment;
FIG. 20 is a diagram illustrating a schematic configuration of a fluorescence inspection apparatus according to a fifth embodiment of the present invention.
FIG. 21 is a diagram illustrating a schematic configuration of a modification of the fifth embodiment;
FIG. 22 is a diagram illustrating a schematic configuration according to a sixth embodiment of the present invention.
[Explanation of symbols]
Reference numeral 1 denotes a sample table, 2 denotes a DNA reaction vessel, 201 denotes a sample tank
202 sample, 3 DNA slide glass reaction vessel
301: sample tank, 302: DNA microarray
4: DNA reaction vessel, 401: sample tank, 402: DNA microarray
5 Objective lens, 5a Optical axis, 6 Base, 7 Fixed axis
701: flange, 7a: screw, 8: bearing
8a: bearing spacer, 8b: C-ring, 9: light source holding member
901: hole, 902: LED light source unit storage hole, 903: screw hole
11 ... LED light source unit, 1101 ... LED light source holder
1102 ... LED light source, 1103 ... Band pass filter
12 O-ring, 13 Spacer, 14 Fan screw, 15 Timing pulley
16 ... motor, 16a ... motor rotating shaft, 17 ... timing pulley
18 timing belt, 19 fixing part, 19a facing surface
20: sliding contact, 21: sensor, 22: filter
23: imaging lens, 24: photodetector, 25: computer
27: Monitor, 28: Fluorescence detection unit
29: LED light source unit driving circuit, 31: LED light source unit
3101: LED light source holder, 3102: LED light source
3103: Bandpass filter, 3104: Diffusion plate
3105: Condensing lens, 32: LED light source unit
3201 ... LED light source holder, 3202 ... LED light source
3203: band pass filter, 3204: diffusion plate
3205: Collimating lens, 33: LED light source unit
3301 ... LED light source holder 3302 ... LED light source
3303: band pass filter, 3304: diffusion plate
34 photodetector 41 41 first LED light source unit 42 42 second LED light source unit
43: Third LED light source unit, 45.46: Dichroic mirror
47: condenser lens, 48: CCD camera, 49: condenser lens
50: CCD camera, 51: condenser lens, 52: CCD camera
53: power supply device, 54: LED light source unit drive circuit,
55: LED light source unit drive circuit, 56: LED light source unit drive circuit
57: AC power supply, 58: Transformer circuit, 59: Bridge rectifier circuit
60: smoothing capacitor, 61.62: power transistor
63: OP amplifier, 64: constant voltage circuit, 65: Zener diode
66 ... OP amplifier, 67 ... variable reference voltage generation circuit
68: variable reference resistor, 69: power switch, 71a to 71e: variable resistor
71.73: Variable resistor, 72a to 72e: Switch
72.74: switch, 81: LED light source unit,
8101: LED light source holder, 8102: LED light source
8103: band-pass filter, 8104: diffusion plate
8105: Condensing lens, 82, 82 ': Reflecting plate, 83: Dichroic mirror
84 optical fiber, 84a light incident end
84b: light emitting end, 91: rotating shaft, 92: bearing, 93: base
94: timing pulley, 95: motor, 95a: motor rotating shaft
96 timing pulley, 97 timing belt

Claims (12)

A sample stage on which the sample is placed,
A semiconductor light source means for irradiating the sample with light and exciting a fluorescent substance contained in the sample,
A condensing lens for condensing at least the fluorescent light emitted when the fluorescent substance is excited by the excitation light, a filter for selectively transmitting the fluorescent light emitted when the fluorescent substance is excited by the excitation light, and the condensing light And a fluorescence detection unit having a photodetector that detects fluorescence that has passed through the lens,
The fluorescence detection apparatus according to claim 1, wherein the sample stage and the semiconductor light source are provided so as to be relatively movable, and irradiates the sample with light from the semiconductor light source at the same time as the relative movement. The semiconductor light source means includes an LED light source that emits excitation light, a filter that selectively transmits a wavelength of light from the LED light source disposed on an optical path of light emitted from the LED light source, a diffusion plate, and a lens. The fluorescence detection device according to claim 1, wherein the fluorescence detection device has all or all of them. 3. The fluorescence detecting device according to claim 1, wherein a plurality of said semiconductor light source means are provided so as to be able to rotate or reciprocate along the periphery of said condenser lens. 3. The fluorescence detecting apparatus according to claim 1, wherein a plurality of said semiconductor light source means are provided so as to rotate or reciprocate along the periphery of said condenser lens, and said rotation or reciprocation is performed an integer number of times. . The irradiation point of light from the semiconductor light source means to the sample is set such that an irradiation point at a peripheral portion of the sample is denser than an irradiation point at a central portion of the sample. Fluorescence detector. 6. A fluorescence detecting apparatus according to claim 3, wherein said semiconductor light source means has substantially the same spectral characteristics of emission wavelengths. 6. The fluorescence detecting device according to claim 3, wherein said semiconductor light source means has a different emission wavelength spectral characteristic. Around the condensing lens, provided rotatably about the optical axis of the condensing lens, and a plurality of the semiconductor light source means are held along the periphery of the condensing lens and emitted from the semiconductor light source means. A light source holding member that holds the semiconductor light source unit at a predetermined angle with respect to the optical axis of the condenser lens so that the sampled light is applied to the sample,
The semiconductor light source unit is provided at a holding portion of the semiconductor light source unit of the light source holding member, and the semiconductor light source unit can adjust at least one of a direction of light emitted from the semiconductor light source unit and a tilt direction with respect to an optical axis of the condenser lens. The fluorescence detection device according to claim 1, further comprising: a position adjustment unit that performs the adjustment. The semiconductor light source unit includes a driving unit that supplies a driving current to the semiconductor light source unit, and the light source holding member includes a power supply unit that supplies a driving current from the driving unit to the semiconductor light source unit. Item 10. The fluorescence detection device according to any one of Items 1 to 8. The light detector includes an image pickup unit, and further has a position detection unit that detects a rotation position of the light source holding member, and determines an optimal exposure time for light detection of the image pickup unit based on an output of the detection unit. The fluorescence detection device according to claim 1, wherein Further, a light intensity detecting means for detecting the intensity of the excitation light to the sample in the vicinity of the sample, and a driving means for supplying a driving current for driving the semiconductor light source means and capable of controlling the magnitude of the driving current. Have
11. The fluorescence detection according to claim 1, wherein the driving current can be controlled by the driving unit based on the intensity of the excitation light detected by the light intensity detecting unit. apparatus. Further, a light intensity detecting means for detecting the intensity of the excitation light to the sample in the vicinity of the sample, and a driving means for supplying a driving current for driving the semiconductor light source means and capable of controlling the magnitude of the driving current. Have
11. The device according to claim 1, wherein a driving current to the semiconductor light source unit can be individually controlled by the driving unit based on an intensity of the excitation light detected by the light intensity detecting unit. Fluorescence detector.

Images