JP2004144839A - Optical scanning device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical scanning device which can read fluorescent information from DNA samples or the like arranged in the form of an array at a high speed by suppressing an unevenness of detection characteristics. <P>SOLUTION: A plurality of optical scanning probes 3A, 3B and 3C incorporating an optical scanning optical system including a condenser lens in an edge part are held with a positioning holding means 4 on an XY scanning stage 5 in order to optically scan a number of sample spots which are specimens arranged in DNA array arranged on a slide glass 2. The positioning means 4 is formed of a voice coil motor roughly movable on an optical axis direction of the condenser lens system and a motion motion actuator minutely movable in a three-axis direction of each optical scanning probe. The fluorescent information can be obtained by performing positioning control maintaining a state for substantially focusing light by each optical scanning probe on the surface of the specimen, and suppressing the unevenness of the detection characteristics. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an optical scanning device for detecting fluorescence from a plurality of sample spots, particularly, microgene spots or microbio spots.
[0002]
[Prior art]
In recent years, with rapid development of genetic research, DNA macroarray technology has emerged as a means for realizing a large amount of gene expression analysis in a short time.
As shown in FIG. 9 in Japanese Patent Application Laid-Open No. 2002-9639 as a first conventional example, a solution containing gene DNA is dropped on a substrate 1 such as a slide glass by several nl each to obtain a DNA microarray. Spot-like spots 2 of about 10 to 100 μm are formed, and these spots are regularly arranged on a single substrate 1 at several thousands to tens of thousands of points.
[0003]
After labeling RNA, which is a specimen, with fluorescence on such a DNA microarray 3 and sprinkling it, washing it, and irradiating each spot 2 with laser light, the fluorescence generated is measured, and gene expression is analyzed based on the fluorescence intensity. be able to.
[0004]
A scanning optical measurement device called a DNA microarray reader is used for the fluorescence measurement in the gene expression analysis. The configuration of this scanning optical measurement device is similar to a general confocal laser microscope, and irradiates a laser beam emitted from a laser light source onto the DNA macro array 3 through an objective lens. The generated fluorescence is guided to a photoelectric conversion element such as a photomultiplier tube (PMT) through a confocal pinhole, and the fluorescence intensity is converted into an electric signal by the photoelectric conversion element.
[0005]
At this time, the DNA microarray 3 is placed on the electric scanning stage and moved in the X and Y directions. Accordingly, the laser light emitted from the laser light source scans the DNA microarray 3 in the X and Y directions, and the electric signal output from the photoelectric conversion element at this time is sent to an image processing device such as a computer. This image processing device performs A / D conversion of an electric signal from a photoelectric conversion element and acquires the signal as scanned image data.
[0006]
As another conventional example of this kind, there is JP-A-2001-108684. In this second conventional example, a multi-spot array light is used to detect a DNA array at high speed.
[0007]
Japanese Patent Application Laid-Open No. 2001-242081 as a third conventional example uses a CD pickup or a pickup having an optical waveguide configuration in order to reduce the size of a DNA array reader.
Also, in Japanese Patent Application Laid-Open No. 2001-238677 as a fourth conventional example, a CD-shaped DNA array is configured for high-speed reading.
[0008]
[Patent Document 1]
JP-A-2002-9639
[0009]
[Patent Document 2]
JP 2001-108684 A
[0010]
[Patent Document 3]
JP 2001-242081 A
[0011]
[Patent Document 4]
JP 2001-238677 A
[0012]
[Problems to be solved by the invention]
In the first conventional example, since a single laser is used for scanning, there is a disadvantage that high-speed reading cannot be performed.
In the second conventional example, the speed can be increased because the multi-spot array light is used. However, since a plurality of spots are generated by using the same optical system, all the spots are accurately positioned on the specimen. If the alignment cannot be carried out as described above, the detection performance will vary greatly. Further, in an optical system that can simultaneously cover a wide range, it is difficult to remove aberrations such as chromatic aberration, and the performance tends to deteriorate depending on the position.
[0013]
In general, it is not possible to handle a plurality of samples whose heights are higher than the micro level and differ. There is a restriction limited to samples on the same slide.
[0014]
In the third conventional example, FIG. 5 in the publication discloses that a plurality of scans are simultaneously performed using a plurality of optical waveguides. However, all spots are accurately positioned on the specimen because they are on the same substrate. If the alignment cannot be carried out as described above, the detection performance will vary greatly. Further, in general, it is not possible to cope with a plurality of samples having different heights on the order of microns or more. There is a restriction limited to samples on the same slide.
The fourth conventional example has a drawback that a special device is required for arranging a sample array with high precision in a spiral shape like a pit of a CD, so that a general matrix DNA array cannot be used. There is a disadvantage that it becomes.
[0015]
(Object of the invention)
The present invention has been made in view of the above points, and provides an optical scanning device capable of reading fluorescence information of an array of DNAs, proteomes, antibodies, cells, and the like at high speed while suppressing variations in detection characteristics. With the goal.
[0016]
[Means for Solving the Problems]
An optical scanning device for detecting fluorescence from a plurality of sample spots, particularly microgene spots or microbio spots,
The sample spot is disposed on a substrate,
A light source,
Light-collecting means for condensing and emitting light from the light source to the test portion,
An optical scanning unit that two-dimensionally scans a focal point condensed on the part to be measured by the condensing unit in a direction orthogonal to an optical axis direction of the condensing unit;
Having a plurality of the light scanning means;
Having light detection means for detecting return light from the test portion,
The optical scanning means has automatic positioning means for performing automatic positioning of at least one degree of freedom, thereby automatically positioning the light scanning means in the optical axis direction of the condensing means and optically scanning the sample spot at a high speed. Fluorescence information from the spot can be read out while suppressing variation in detection characteristics.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(First Embodiment)
1 to 10 relate to a first embodiment of the present invention. FIG. 1 shows an overall configuration of an optical scanning device according to the first embodiment, and FIG. 2 shows optical scanning optics on the distal end side of an optical scanning probe. 3 shows the configuration of the scanner in FIG. 2, FIG. 4 shows a coarse actuator for positioning and holding the probe unit, and FIG. 5 shows the configuration of a fine actuator provided near the base end of the optical scanning probe. FIG. 6 shows a state of a scanning method using sample spots arranged on a DNA array, FIG. 7 shows an example of a process of positioning a three-dimensional position before actually performing optical scanning, and FIG. FIG. 9 shows a case in which different DNA arrays are scanned by an optical scanning probe, FIG. 9 shows a state of the scanning method using sample spots arranged on the DNA array in the case of FIG. 8, and FIG. And positioning and holding the scanning probe showing a configuration of a main part of the case of performing optical scanning.
[0018]
As shown in FIG. 1, an optical scanning device 1 according to the first embodiment of the present invention includes, for example, a substrate on a slide glass 2 and a small gene spot (hereinafter referred to as a sample spot) disposed on, for example, a DNA array 55 described later. ), Optical scanning probes 3A, 3B, and 3C each including a built-in optical scanning unit for optically scanning a minute scanning range (observation range) in detail, and these optical scanning probes 3A, Positioning and holding means 4 for positioning and holding the 3B and 3C, and an electric motor as coarse movement scanning means for two-dimensionally coarsely scanning the optical scanning probes 3A, 3B and 3C attached via the positioning and holding means 4. An XY scanning stage 5, an XY scanning stage driving device 6 for driving the XY scanning stage 5, and optical scanning probes 3A, 3B, and 3C are connected to supply light from a light source. At the same time, detection units 7A, 7B, and 7C each having a detection unit for detecting a fluorescent component and the like in the return light from the test object side, and are connected to these detection units 7A, 7B, and 7C, and For example, a personal computer (hereinafter abbreviated as PC) 8 as image processing means for performing image processing on a signal, a monitor 9 for displaying an optical scanning image formed by the PC 8, and recording optical scanning image data and the like. (Storage).
[0019]
The optical scanning device 1 reads fluorescence information for the DNA array 55 shown in, for example, FIGS. In this case, a two-fluorescent labeling method is employed. The principle is to detect the difference in gene expression between two different mRNA samples, each of which is labeled with a different fluorescent dye and competitively hybridized (amplified) on the DNA array 55 to detect both fluorescence. And compare. In this case, one or more excitation lights are generally used, and the amounts of fluorescence are compared using detectors corresponding to the fluorescence wavelengths of the respective fluorescent dyes.
[0020]
In order to perform such a detection operation, the detection unit 7A includes an Ar laser 11a, a He-Ne laser 11b, and a semiconductor laser (laser laser) so that a plurality of, for example, three wavelengths of laser light can be generated as excitation light sources. These laser beams are combined by dichroic mirrors (or herb mirrors) 12a, 12b, and 12c, are incident on a condenser lens 13, and are condensed by the condenser lens 13 to form an optical fiber 14a. Is incident on.
[0021]
A part of the laser light incident on the optical fiber 14a is guided by the optical coupler 15 to the optical fiber 14b, and the light guided to the optical fiber 14b passes through the optical connector 16 and is transmitted to the optical scanning probe 3A. The light enters the fiber 17a.
[0022]
The light incident on the optical fiber 17a is transmitted to the distal end side. The distal end of the optical fiber 17a is inserted into the optical scanning probe 3A, and an optical scanning optical system 19 is provided at the distal end. This configuration is shown in FIG.
[0023]
As shown in FIG. 2, the optical scanning probe 3A has a rigid distal end frame 22 constituting a distal end portion 21 connected to the distal end of a rigid sheath 20, and the distal end side of the optical fiber 17a has a micromachine mirror inside the distal end frame 22. Together with a scan mirror (also referred to as a scanner) 23 for two-dimensional (for example, XY) scanning by the mounting member 18 on the inner wall of the end frame 22.
[0024]
The light emitted from the very small distal end face 24 of the optical fiber 17a is reflected by a fixed mirror 25 fixed to the inner wall surface of the distal end frame 22, further reflected by the movable portion of the scanner 23, and The light is emitted in the direction of the optical axis O of the condenser lens system 26 through the condenser lens system 26 which is disposed and attached to the opening of the end frame 22, and is condensed at the focal point 27 at this time. Irradiated on the side.
In this case, the movable mirror in the scanner 23 is two-dimensionally tilted, so that the focal point 27 is two-dimensionally scanned (moved) in a direction orthogonal to the optical axis O.
[0025]
The reflected light from the focal point 27 follows the same path in reverse, and is incident on the distal end face 24 of the optical fiber 17a. That is, the distal end face 24 and the focal point 27 of the optical fiber 17a have a confocal relationship or a relationship close thereto (near confocal relationship) with respect to the optical scanning optical system 19, that is, the fixed mirror 25, the scanner 23, and the condenser lens system 26. Only light near the focal point 27 is incident on the distal end surface 24 of the optical fiber 17a.
[0026]
Note that, in actuality, the fluorescent light emitted from the focal point 27 and the vicinity thereof is mainly detected. Of course, the reflected light component is also detected, and the intensity of the reflected light is detected so that the fluorescence can be detected (by normalization or the like) in a comparable manner, and also used for controlling the optical scanning while maintaining the focus on (or close to) the subject. Like that.
[0027]
Note that an achromatic lens is employed as the condenser lens system 26 in the present embodiment. That is, a plurality of lenses are combined so as to have almost the same focal position with respect to the light of the light source, that is, the light of each wavelength of the Ar laser 11a, the He-Ne laser 11b, and the laser diode 11c and the wavelength of the fluorescence described later. Is formed.
[0028]
The scanner 23 is connected to a scanner driving device 30 inside the detection unit 7A via a signal line 28a and further via a connector 29a, and outputs a driving signal for scanning in the X and Y directions from the scanner driving device 30 to move the scanner 23. The mirror part tilts in the X and Y directions, two-dimensionally scans the focal point 27 focused on the subject side, and obtains information for analyzing gene expression of the subject by the returned light. I have.
Hereinafter, the configuration of the scanner 23 will be described with reference to FIGS.
The scanner 23 includes a gimbal mirror 32 serving as a movable mirror and a ground 34 having a concave portion 33.
[0029]
The main body of the gimbal mirror 32 is a silicon plate, and the signal line 28a shown in FIG. 2 is actually formed by driving wirings 35a, 35b, 35c, and 35d as shown in FIG. 36b, 36c, and 36d. Here, the driving wiring and the electrode also serve as a mirror.
A satin portion 37 in FIG. 3 is an opening, and a central mirror portion is rotatable in X and Y directions around hinge portions 38 and 39.
[0030]
The electrodes 36a, 36b and 36c, 36d are respectively connected to the scanner driving device in the detection unit 7A via a pair (two) of X-direction driving wires 35a, 35b and a pair (two) of Y-direction driving wires 35c, 35d. 30 are electrically connected to each other, and an X drive signal and a Y drive signal are supplied, respectively.
[0031]
A part of the light incident on the optical fiber 17a is guided to the optical fiber 14c by the optical coupler 15 in the detection unit 7A. The light guided to the optical fiber 14c is emitted from the end face, and is condensed by the condensing lens 41 constituting the light detection unit 40A.
The end of the optical fiber 14d is closed or treated so that reflection from the end is attenuated.
[0032]
The light condensed by the condenser lens 41 is split into, for example, reflected light and transmitted light by a half mirror 42. Then, the transmitted light is detected by a plurality of detecting means for detecting different predetermined wavelengths, and the reflected light is reflected by the return light (mainly reflected light) which detects return light from the subject. It is detected by the light detecting means.
[0033]
That is, a dichroic mirror 43a that transmits light of the wavelength λ1 and reflects the other light is disposed opposite the half mirror 42, and the light transmitted through the dichroic mirror 43a is a photomultiplier (abbreviated as PMT) 44a. After being photoelectrically converted by optical doubling, the signal is converted from an analog signal to a digital signal by the A / D converter 45a and input to the PC 8.
[0034]
A dichroic mirror 43b that reflects light of the wavelength λ2 and transmits the other light is disposed on the reflection optical path side of the dichroic mirror 43a. The light reflected by the dichroic mirror 43b is incident on the PMT 44b, After being photoelectrically converted by doubling, the signal is converted into a digital signal by the A / D converter 45b and input to the PC8.
[0035]
A dichroic mirror 43c that reflects light of wavelength λ3 and transmits the other light is disposed on the transmitted light path side of the dichroic mirror 43b. The light reflected by the dichroic mirror 43c is incident on the PMT 44c, After being photoelectrically converted by doubling, the signal is converted into a digital signal by an A / D converter 45c and input to the PC 8.
[0036]
On the other hand, the light reflected by the half mirror 42 is received by a photodetector (abbreviated as PD) 46, photoelectrically converted, and then converted from an analog signal to a digital signal by an A / D converter 45 d and input to the PC 8. You.
[0037]
In the PC 8, the signal intensity of each of the wavelengths λ1 to λ3 detected via the A / D converters 45a to 45c is standardized by the intensity of the signal (from the photodetector 46) converted by the A / D converter 45d. To detect.
The other optical scanning probes 3B and 3C have the same structure as the optical scanning probe 3A, and the detection units 7B and 7C have the same structure as the detection unit 7A.
[0038]
As shown in FIG. 4, the probe unit 3 including the optical scanning probes 3A, 3B and 3C has a rear end attached to an XY scanning stage 5 via a positioning and holding means 4, and the XY scanning stage 5 performs optical scanning. The probes 3A, 3B and 3C are moved (scanned) in the X direction and the Y direction as a whole, that is, for each probe unit 3.
[0039]
As will be described later, the scanning range in the X and Y directions by each optical scanning probe 3I (I = A, B, C) is narrow. It moves in the direction and the Y direction to cover a predetermined inspection range.
[0040]
In this case, each optical scanning probe 3I is formed with a fine movement actuator 51I (only 51A is shown in FIG. 4) as a fine movement positioning means near the base end thereof, and the base end of the fine movement actuator 51I is further integrated. The XY scanning stage 5 is attached to, for example, a Y stage portion 5b via, for example, a voice coil motor 52 as coarse movement positioning means in the Z direction.
[0041]
The Y stage portion 5b is held on the bottom surface of the X stage portion 5a movable in the X direction along the guide rod 53, and the Y stage portion 5b is fitted in a guide groove (not shown) provided on the bottom surface of the X stage portion 5a. In this case, it is held movably in the Y direction.
The X stage 5a and the Y stage 5b are moved in the X and Y directions by a motor (not shown), respectively. Each motor is driven by a driving signal from the XY scanning stage driving device 6 in FIG.
[0042]
For example, a DNA array 55 as a base material on which a test object is formed is arranged on the slide glass 2 arranged to face the probe unit 3, and all the sample spots 65 on the DNA array 55 (see FIG. 6) In this case, excitation light is irradiated by a combination of two-dimensional (XY) coarse movement scanning by the XY scanning stage 5 and two-dimensional (XY) light scanning by the probe unit 3 so that fluorescence can be detected at that time. ing.
[0043]
FIG. 4 schematically shows an observation range 57i (i = a to c) that can be observed by scanning with each optical scanning probe 3I.
When the sample spot 65 is optically scanned by each optical scanning probe 3I, it is necessary to maintain the light irradiated on the sample spot 65 in a state close to a focus state where a focus is formed on the sample surface. In the embodiment, the positioning and holding means 4 by the fine movement actuator 51I and the voice coil motor 52 is provided as described above.
[0044]
In this case, the approximate positioning in the Z-axis direction which is the optical axis direction (of the condenser lens system 26 forming the optical scanning optical system 19) is performed on the entire optical scanning probes 3A to 3C by the common voice coil motor 52. The precise positioning of the individual optical scanning probes 3A to 3C in the Z direction can be performed independently by the fine movement actuators 51A to 51C, so that high-precision positioning can be performed.
[0045]
FIG. 5 shows the structure of the fine movement actuator 51A provided at the base end (rear end) of the optical scanning probe 3A. FIG. 5 (A) shows the structure when viewed from the Y direction, and FIG. 5 (B) shows the structure when viewed from the X direction. Is shown. The other fine movement actuators 51B and 51C have the same configuration as fine movement actuator 51A.
[0046]
The fine movement actuator 51A is disposed in such a manner that two plate-shaped bimorph piezoelectric elements 61 and 62 for scanning in the Y and X directions are opposed to each other in the Y and X directions in the longitudinal direction of the base end of the optical scanning probe 3A. And in the X direction.
[0047]
Further, a laminated piezoelectric element 63 having a laminated structure for scanning in the Z direction is attached to the rear end of the plate-shaped bimorph piezoelectric element 62 for scanning in the X direction, and is capable of expanding and contracting in the Z direction. The rear end of the laminated piezoelectric element 63 is attached to a movable portion of the voice coil motor 52, for example, a permanent magnet, and a voice coil serving as a fixed portion around the permanent magnet is attached to the Y stage portion 5b.
[0048]
The voice coil motor 52 and the fine actuator 51A are connected to, for example, the PC 8 so that the PC 8 can control the operations of the voice coil motor 52 and the fine actuator 51A.
[0049]
For example, by inputting a movement operation from a keyboard or the like provided on the PC 8, the holding position in the Z direction by the voice coil motor 52 is adjusted by the input value, or the fine scanning actuator 51A is also scanned with light. The base position of the probe 3A is moved in the X, Y, and Z directions, so that the positioning position can be finely adjusted three-dimensionally.
[0050]
Further, the PC 8 adjusts the holding position of the laminated piezoelectric element 63 so that the luminance level becomes maximum based on the output signal from the photodetector 46.
For example, as shown in FIG. 4, the optical scanning probes 3A to 3C are attached, and the PC 8 displaces the laminated piezoelectric element 63 of the fine movement actuator 51A in the Z direction at an appropriate representative point in the DNA array 55 arranged on the slide glass 2. A drive signal is applied so that the luminance level (that is, the reflected light component) is detected from the output signal from the photodetector 46, and the amplitude value of the drive signal at the position where the luminance level becomes the highest is stored in the internal signal. It is stored in storage means or the like.
[0051]
When the DNA array 55 is actually optically scanned, the holding position of the laminated piezoelectric element 63 in the Z direction is controlled by, for example, interpolating the stored information (that is, the amplitude value of the drive signal) at the representative point. Optical scanning of the DNA array 55 can be maintained in a substantially focused state.
[0052]
Further, in the present embodiment, since each optical scanning probe 3I is held independently and finely adjustable in three axial directions by the fine movement actuator 51I, the optical scanning optical system on the distal end side of each optical scanning probe 3I. 19 and the DNA array 55 to be observed can be set to an appropriate state in which optical information can be read by optical scanning.
[0053]
For example, the optical scanning probe 3A is controlled so as to be automatically positioned so that light from each of the optical scanning optical systems 19 incorporated therein substantially focuses on the surface of the subject in the optical axis O direction. The same applies to the other optical scanning probes 3B and 3C.
[0054]
FIG. 6 shows a two-dimensional array of sample spots 65 formed on the DNA array 55. In these sample spots 65, for example, an observation range 57a indicated by a thick solid line is an optical scanning probe 3A, and an observation range 57b is an optical scanning probe. The light is read by the optical scanning probe 3B adjacent to the probe 3A.
An observation range (not shown) adjacent to the right side of the observation range 57b (shown as 57c) is read by the optical scanning probe 3C.
[0055]
Then, the observation ranges 57a, 57b, and 57c are read by the three optical scanning probes 3A to 3C, respectively. Thereafter, the entire optical scanning probes 3A to 3C are slightly moved, for example, in the vertical direction (for example, the lower side in FIG. 6) by the XY scanning stage 5, for example, by the moving amount Y1 indicated by the arrow, and the three optical scanning probes 3A are similarly moved. Each observation range is read at ~ 3C. In FIG. 6, the dotted line indicates that the observation range 57a is also moved by the movement of the optical probe 3A by the movement amount Y1, and becomes the reference numeral 57a '.
[0056]
When the entire sample spot 65 in the vertical direction is read by gradually moving in the vertical direction in this way, the XY scanning stage 5 returns the sample spot 65 to the initial position, and then the entire optical scanning probe 3A to 3C is moved in the horizontal (horizontal) direction , For example, is moved by a movement amount X1 indicated by an arrow, and the observation range is similarly read by each of the three optical scanning probes 3A to 3C. Thereafter, the moving range is slightly moved downward, and the three optical scanning probes 3A to 3C are moved. Are used to read the observation range. In this manner, all the sample spots 65 are optically scanned by the three optical scanning probes 3A to 3C.
[0057]
In this case, the return light from each sample spot 65 within the observation range 57i is guided into the detection unit 7I, and received by the photodetector 46 and the PMTs 44a to 44c. Before the PMTs 44a to 44c, selective wavelength separation means (dichroic mirrors 43a to 43c) can separate and extract only the respective wavelength components of the wavelengths λ1 to λ3 so that they are incident. Fluorescence can be detected.
[0058]
The signals that have been photoelectrically converted and doubled by the PMTs 44a to 44c are A / D converted and input to the PC 8, and are detected by the photodetector 46 based on the ratio with the A / D converted signals. The intensity is normalized and detected.
[0059]
The detected fluorescence intensity data is stored in a storage device such as a hard disk in the PC 8 and is also stored in the memory, and an appropriate amount of data is displayed on the monitor 9 or stored (stored) in the data storage device 10. You can also do it.
[0060]
In addition, as shown in FIG. 6, the movement range Y1 is set so that the observation ranges 57a and 57a 'partially overlap each other, so that a small variation at the time of coarse movement can be covered. The same applies to the horizontal movement amount X1.
[0061]
In addition, since the light scanning probes 3A and 3B and 3B and 3C detect fluorescence in the same part by overlapping a part in the horizontal direction, it is also possible to correct variations in their characteristics. . In other words, it is possible to correct variations in characteristics including the detection unit 7I between the adjacent optical scanning probes 3A and 3B, 3B and 3C, and perform highly accurate detection in which variations in characteristics are corrected.
Although the operation has been described above together with the configuration of the present embodiment, the positioning operation of the present embodiment will be described with reference to FIG.
[0062]
First, as shown in step S1, the rear end side of the probe unit 3 is attached to the XY scanning stage 5 via the positioning and holding means 4.
Further, the subject side is set as shown in step S2. Specifically, the DNA array 55 is set on the slide glass 2.
[0063]
Then, as the next step S3, three-dimensional positioning adjustment between the XY scanning stage 5 to which the probe unit 3 is attached and the subject side is performed. As this positioning adjustment, for example, the three-dimensional position of the distal end surface of the probe unit 3 with respect to the subject is manually set near a predetermined position. For example, in FIG. 6, it is set near an initial position (indicated by P0 in FIG. 6) where the optical scanning probe 3A performs optical scanning, and is set near a distance such that a focus state is established at the position P0.
[0064]
Next, a focus adjustment instruction is input from a keyboard or the like (not shown) connected to the PC 8, and the PC 8 starts a positioning operation for automatic focus adjustment by this input. Specifically, the PC 8 sends a drive signal to the voice coil motor 52 to move the movable part of the voice coil motor 52, and monitors the luminance level detected by the optical scanning probe 3A via the light detector 46. Then, the Z-direction adjustment is performed so that the position where the value becomes the maximum is determined as the focus state, and the position is adjusted at that position. Further, the XY scanning stage 5 performs the positioning so that light strikes the initial position.
[0065]
By this adjustment, the other optical scanning probes 3B and 3C are set to a state close to the focus state, and are set to positions close to the initial positions, respectively. Therefore, the other optical scanning probes 3B and 3C can be positioned by fine adjustment by the fine movement actuators 51B and 51C.
[0066]
For this reason, for example, in the optical scanning probe 3B, the fine movement actuator 51B is further driven to adjust the positioning in the Z direction so as to be in the focus state and to position the light so as to impinge on the initial position of the observation range 57b. Further, the fine scanning actuator 51C also performs positioning in the Z direction so that light is applied in the focused state to the optical scanning probe 3C, and also allows light to be applied to the initial position of the observation range 57c.
[0067]
Thus, three-dimensional positioning of the three optical scanning probes 3A to 3C is performed.
As described above, each optical scanning probe 3I is independently held by at least the fine movement actuator 51I so as to be adjustable with three degrees of freedom. Therefore, it is possible to position each of the optical scanning probes 3I so that the light strikes the initial position or the like in a focused state. it can.
[0068]
In the next step S4, the driving voltage data of the laminated piezoelectric element 63 at the time of moving to the appropriately set representative point in each observation range and focusing on each representative point is acquired and stored.
[0069]
As representative points in this case, for example, several points are set in each observation range, drive voltage data in a state where each representative point is in focus is obtained, and interpolated drive voltage data is obtained in a portion other than those representative points. To make it almost in focus. Therefore, when the difference in the value of the drive voltage data between the representative points is large, the number of the representative points may be increased. Then, the drive data for focusing shown in step S5 is generated by, for example, interpolating the stored drive voltage data according to the position during scanning.
[0070]
Then, when the sample spot 65 of the DNA array 55 is actually optically scanned from the initial position as shown in step S6, focus control is performed using the focus driving data.
[0071]
That is, the laminated piezoelectric element 63 is driven by the driving data for focusing, and the light irradiated by each optical scanning probe 3I is automatically controlled so as to keep almost the state of being focused on the sample spot 65. Gene information can be obtained by detecting with the light detection unit 40I of the detection unit 7A.
[0072]
As described above, in the present embodiment, focus control (positioning in the Z-axis direction) is performed in an open loop so that light can be focused on the sample spot 65 almost in a focused state. However, focus control is performed in a closed loop as described later. It may be performed.
[0073]
As described above, in the present embodiment, a plurality of optical scanning probes 3I can be used to simultaneously perform optical scanning, respectively, so that a desired fluorescence intensity can be detected at high speed.
[0074]
In this case, as described above, the positioning in the longitudinal direction of each optical scanning probe 3I, that is, the optical axis direction of the condenser lens 26 (distance direction facing the sample spot 65 side) can be controlled independently. By setting each of the optical systems almost in the focused state, the sample spot 65 can be optically scanned, the variation in the detection characteristics can be suppressed, and detection can be performed. Further, a focus error, that is, a detection omission due to defocus can be prevented.
[0075]
Further, in the present embodiment, light of a plurality of wavelengths is generated as an excitation light source so as to be able to irradiate the sample spot 65, so that the fluorescence wavelength to be detected can be efficiently excited, S / N can be improved.
[0076]
In the above description, the case where one DNA array 55 is arranged on the slide glass 2 and three optical scanning probes 3A to 3C perform optical scanning to detect fluorescence has been described. As shown, for example, DNA arrays 55A, 55B, and 55C may be arranged on each of the three cover glasses 2, and three optical scanning probes 3A to 3C may perform optical scanning to detect fluorescence.
In this case, the manner in which the sample spots 65 of the DNA arrays 55A, 55B, and 55C are optically scanned is as shown in FIG.
[0077]
Also in this case, in the present embodiment, the three optical scanning probes 3A to 3C can be positioned and held independently of each other, so that the sample spots of the DNA arrays 55A, 55B, and 55C are similar to the case of FIG. 65 can be optically scanned in a focused state to detect fluorescence.
[0078]
In the above description, three optical scanning probes 3A to 3C have been described. However, as shown in FIG. 10, a probe unit 3 formed two-dimensionally using a plurality of optical scanning probes 3A to 3D,. It may be attached to the XY scanning stage 5 via a voice coil motor 52 as a coarse movement positioning means. In FIG. 10, one DNA array 55 is shown to be optically scanned, but a plurality of DNA arrays 55A, 55B, 55C,... As shown in FIG.
[0079]
As described above, according to the present embodiment, the subject is scanned by the plurality of optical scanning probes 3A to 3C and the like, so that fluorescence information can be obtained at high speed and the optical scanning of each optical scanning probe can be performed. Since automatic control is performed independently and independently so as to focus the state of focusing on the object by the optical system, variations in detection characteristics can be prevented or suppressed.
[0080]
In the above description, the focus control is performed in an open loop, but may be performed in a closed loop. For example, a signal that has been photoelectrically converted so that the amount of light detected by the photodetector 46 is maximized is compared with a previously detected signal level, and the signal of the difference controls the driving of the laminated piezoelectric element 63 of the fine actuator 51I. In such a case, the focus state is set first.
[0081]
Further, focus control is performed in a closed loop in a portion other than the sample spot 65 in this way, and control is performed so that the focus control state immediately before the sample spot 65 is maintained (for example, focus control in a sample-hold state). You may do it.
[0082]
That is, since the surface of the DNA array 55 other than the sample spot 65 can be made uniform, the focus of the closed pool is controlled in this portion, and the amount of reflected light is controlled by the optical characteristics (reflection characteristics) of the surface in the sample spot 65 portion. Since it may change (from the case of the surface of the DNA array 55), the sample spot 65 may be performed in the focus control state immediately before (that is, in the held control state).
[0083]
In order to perform focus control in a partially closed loop as described above, it is preferable that the surface portion of the DNA array 55 be easily distinguished from the sample spot 65. For example, two photodetectors are provided on the side of the photodetector 46 for detecting the reflected light so as to selectively receive two wavelengths of light so that a wavelength matched to the surface color of the DNA array 55 can be identified. Is also good. Then, the output signals of the two photodetectors may be used to switch between the focus control in the closed loop and the held focus control.
[0084]
In the above description, the DNA array 55 for detecting and quantifying DNA and RNA is described as the sample spot 65. However, the present invention is not limited thereto, and an antibody array for detecting an antibody-antigen reaction by fluorescence, an antibody-antigen The present invention can be applied to an array of a protein bioarray chip utilizing a specific molecular recognition mechanism such as a reaction, an enzyme-substrate, a receptor-ligand, and a protein-DNA.
[0085]
(Second embodiment)
Next, a second embodiment of the present invention will be described. FIG. 11 shows a configuration of a main part of an optical scanning device 1B according to a second embodiment of the present invention. In the first embodiment, an excitation light source is prepared for each of the optical scanning probes 3A to 3C. However, the optical scanning device 1B is configured to use only a common set of excitation light sources.
[0086]
That is, the light of one set of excitation light sources 70 is incident on the optical fiber 71a by the condenser lens 13, and the light is guided to the optical fibers 71b and 71c by the optical coupler 72a. One set of excitation light sources 70 includes the Ar laser 11a, the He-He laser 11b, the laser diode 11c, the dichroic mirrors 12a, 12b, 12c, and the condenser lens 13 shown in FIG.
[0087]
The light guided to the optical fiber 71b is guided to the optical fibers 71d and 71e by the optical coupler 72b. Further, the light guided to the optical fiber 71c is also guided to the two optical fibers 71f and 71g by the optical coupler 72c.
[0088]
The light guided to the optical fiber 71d is guided by the optical coupler 72d to the optical fibers 71h and 71i, respectively, and the light guided to the optical fiber 71h passes through the optical connector 16a to the optical fiber 17a of the optical scanning probe 3A. Light is guided. The end of the optical fiber 17i is closed.
[0089]
Similarly, the light guided to the optical fiber 71e is guided to the optical fibers 71j and 71k by the optical coupler 72e, and the light guided to the optical fiber 71j is transmitted to the optical scanning probe 3B via the optical connector 16b. Is guided to the optical fiber 17b. The end of the optical fiber 17k is closed.
[0090]
The light guided to the optical fiber 71f is guided by the optical coupler 72f to the optical fibers 71l and 71m, respectively, and the light guided to the optical fiber 71l passes through the optical connector 16c and is connected to the optical fiber of the optical scanning probe 3C. The light is guided to 17c. The end of the optical fiber 17m is closed.
Similarly, the light side guided to the optical fiber 71g has the same configuration.
[0091]
The return light from the subject side by the optical scanning probe 3A is incident on the optical coupler 72d via the optical fiber 71h, a part of which is guided to the optical fiber 71o, and is incident on the photodetector 40A.
Also, the return light from the subject side by the optical scanning probe 3B is incident on the optical coupler 72e via the optical fiber 71j, and a part thereof is guided to the optical fiber 71p and is incident on the photodetector 40B.
[0092]
The return light from the subject side by the optical scanning probe 3C enters the optical coupler 72f via the optical fiber 71l, a part of which is guided to the optical fiber 71q, and enters the photodetector 40C.
Each of the optical scanning probes 3A, 3B, 3C,... Has a signal line 28i extending therefrom and connected to the scanner driving device 30, but is omitted in FIG. 11 for simplicity.
[0093]
Other configurations are the same as those of the first embodiment. The operation (operation) according to the present embodiment is almost the same as that of the first embodiment except that the light source 70 is shared.
According to the present embodiment, the cost can be reduced because only one set of the light sources 70 is required.
[0094]
Further, as shown in FIG. 2, the optical scanning optical system 19 such as the optical scanning probe 3A can be composed of the fixed mirror 25, the scanner 23, and the condenser lens system 26. In the first or the present embodiment, for example, FIG. An optical scanning optical unit 75 as shown in FIGS. 12 and 13 may be employed.
[0095]
The optical scanning optical unit 75 shown in FIGS. 12 and 13 is configured to two-dimensionally scan the condenser lens system 26 in a direction orthogonal to the optical axis thereof together with the tip of the optical fiber 17a. It is unnecessary.
As shown in FIG. 12, the optical scanning probe 3A has a distal end 21 formed by fixing an optical frame 78 having a built-in optical scanning optical unit 75 inside the outer sheath 76 at the distal end thereof.
[0096]
In addition, a cover glass 88 is attached to a distal end surface of the optical frame 78 in which the optical scanning optical unit 75 is built via a distal end cover holder 77, and the distal end opening of the optical frame 78 is sealed in a watertight manner.
The optical scanning optical unit 75 has a base 79 fixed to an optical frame 78. The base 79 is set to be heavier than the lens frame 84 and the condensing lens system 26 for condensing so as not to move easily. ing.
[0097]
The vicinity of the tip of the optical fiber 17a is fixed to the base 79. As shown in FIG. 13, the rear end of the thin plate 80 is adhered to both sides of the base 79. The piezoelectric element 74 polarized in the thickness direction is bonded to the thin plate 80. A signal line 28 a for driving the piezoelectric element 74 is connected to the piezoelectric element 74. This signal line 28a passes through the inside of the optical scanning probe 3A and is connected to the scanner driving device 30 via the connector 29a in FIG.
[0098]
The tip of the thin plate 80 is fixed to the relay member 81. Parallel thin plates 82a and 82b are fixed to the rear ends of the relay member 81. Piezoelectric elements 83a and 83b are bonded to the thin plates 82a and 82b.
A lens frame 84 is fixed to the distal ends of the thin plates 82a and 82b, and a ferrule 86 that fixedly holds the condenser lens system 26 and the distal end of the optical fiber 17a is fixed to the lens frame 84.
[0099]
After the optical fiber 17a is fixed to the ferrule 86, the distal end surface 24 is polished integrally with the ferrule 86, and an antireflection film is further provided. Further, the piezoelectric elements 83a and 83b are connected to the scanner driving device 30 via a signal line 28a.
[0100]
Further, in the present embodiment, a small displacement sensor 86 is adhesively fixed to the lens frame 84, and is connected to the scanner driving device 30 via the signal line 28a. Then, the displacement sensor 86 outputs, for example, a voltage that maximizes the scanning position in the X direction of the scanner at the farthest position, that is, the position at the right end of the display image, based on the left end of the display image on the monitor 9. Then, the amplitude of the drive signal by the drive signal generator in the scanner drive device 30 is adjusted.
With such a structure, the distal end portion 21 of the probe 3A is sealed, so that the focal point 27 can be two-dimensionally scanned in a direction orthogonal to the optical axis O by optical scanning.
[0101]
Although the above-described optical scanning means performs two-dimensional scanning using a piezoelectric element, even in the case of the gimbal mirror structure employed in the first embodiment, a hole is provided at the center of the ground in the gimbal mirror structure. Is provided to fix the distal end of the optical fiber 17a, so that the light is two-dimensionally transmitted almost in the same manner as described in the first or second embodiment without using the fixed mirror 25. A scanning structure can be used. This content is disclosed in FIGS. 16 to 19 in JP-A-2001-076696.
[0102]
(Third embodiment)
Next, a third embodiment of the present invention will be described. In the present embodiment, fluorescence detection is performed using a microbio spot as a sample spot, and is an apparatus for obtaining desired information by optically scanning a large number of sampled tissue sections at high speed.
More specifically, a tissue specimen array in which hundreds of tissue sections are mounted on one slide is prepared, and function-specific staining such as tissue immunostaining can be performed on many specimens at once. Since a large number of almost identical slides can be obtained from the same specimen, various stainings can be performed on many specimens.
[0103]
FIG. 14 shows the configuration of the peripheral portion of the optical probe unit according to the third embodiment. In FIG. 14, a tissue sample array 91 is arranged in place of the DNA array 55 in FIG. 10, and a tissue section 92 arranged in the tissue sample array 91 is optically scanned to detect fluorescence from each of them, and the fluorescence is detected. The information added or associated with the information can be obtained. In FIG. 14, observation ranges 57a, 57b and the like are indicated by the same reference numeral 57 in a simplified manner.
The method for manufacturing the tissue specimen array 91 and the method for fluorescent labeling in this case will be described with reference to FIG.
[0104]
FIG. 15A shows a plurality of small-diameter pipes 93 to be sampled, and as shown in FIG. 15B, a plurality of pipes 93 are driven into the tissues 94A and 94B to be examined. Then, pipes A1, A2, A3 and B1, B2, B3 each containing a cylindrical structure having an appropriate thickness in each pipe 93 are formed. In addition, a similar pipe from another organization (not shown) is formed.
[0105]
Then, as shown in FIG. 15 (C), these pipes A1, A2, A3,... Are fixed in a state of being bundled with an adhesive (not shown) or the like, and then sliced thinly with a cutting machine or the like, and the tissue section 92A or A large number of tissue specimen arrays 91 in which 92B (represented simply by reference numeral 92 in FIG. 14) are arranged can be prepared (prepared).
[0106]
Then, in the tissue specimen array 91 prepared in this way, for example, the tissue specimen array 91A shown in FIG. 15 (D) which has been subjected to fluorescent immunostaining of p53 protein is placed on the cover glass 2 and optically scanned as shown in FIG. Thus, p53 protein fluorescent immunological information for each tissue section 92 can be obtained.
[0107]
Further, as shown in FIG. 15 (E), a tissue specimen array 91B processed by FISH (Fluorescence in situ hybridization) so as to detect K-ras gene expression is placed on the cover glass 2, and the first or second embodiment is performed. The corresponding information can be obtained by performing optical scanning with the optical scanning device described in the embodiment.
[0108]
In the above, the case of the tissue specimen array 91 for the cell section 92 has been described, but the present invention may be applied to the case of a cell culture container 96 as shown in FIG.
[0109]
As shown in FIG. 16, minute cells 97 are arranged in a cell culture vessel 96 in a 12-dimensional manner, and fluorescence sampling is performed on the cells cultured in each cell 97, and the optical scanning described in the first or second embodiment is performed. Optical scanning with the device can also provide information related to fluorescence sampling.
[0110]
In the above description, the probe unit 3 side is attached to the XY scanning stage 5 and the probe unit 3 side is roughly moved, but the subject side on which the DNA array 55 is arranged is moved by the XY scanning stage 5. You may make it coarsely move.
[0111]
For example, in FIG. 6, after scanning the observation ranges 57 a, 57 b, and 57 c on the probe unit 3 side, instead of moving the probe unit 3 by the XY scanning stage 5 to the lower side in FIG. It is apparent that the movement control may be performed such that the slide glass 2 side on which is disposed is moved upward by the movement amount Y1 in FIG.
[0112]
Note that the optical scanning probe 3I shown in FIG. 4 or the like may have a shorter sheath length or a portion near the base end of the distal end portion 21 in FIG. 2 may be the rear end of the optical scanning probe. In this case, in FIG. 4 or FIG. 5, the fine movement actuator 51I for performing minute (high-definition) positioning in three axial directions is provided at the rear end of the optical scanning probe 3I, but near the front end side (for example, the base end of the front end portion). (Nearby).
Note that embodiments and the like configured by partially combining the above-described embodiments and the like also belong to the present invention.
[0113]
[Appendix]
1. An optical scanning device for detecting fluorescence from a plurality of sample spots, particularly microgene spots or microbio spots,
The sample spot is disposed on a substrate,
A light source,
Light-collecting means for condensing and emitting light from the light source to the test portion,
An optical scanning unit that two-dimensionally scans a focal point condensed on the part to be measured by the condensing unit in a direction orthogonal to an optical axis direction of the condensing unit;
Having light detection means for detecting return light from the test portion,
Having a plurality of the light scanning means;
An optical scanning device, comprising: an automatic positioning unit that performs automatic positioning of the optical scanning unit with at least one degree of freedom.
[0114]
1.1. In Appendix 1, one degree of freedom moves in the optical axis direction.
1.1.1. In Appendix 1.1, the automatic positioning means makes the focused focal point substantially coincide with the sample surface.
1.1.1.1. Appendix 1.1.1, wherein the optical scanning means is driven by the automatic positioning means at a position where the reflected light from the sample is maximized.
[0115]
1.1.1.2. In the supplementary note 1.1.1, there is provided a fine movement positioning means which moves in a direction perpendicular to the optical axis direction.
1.2. In Supplementary Note 1, the plurality of optical scanning units have independent automatic positioning units each having at least one degree of freedom.
1.3. In Appendix 1, a plurality of optical scanning means reads a sample spot on a single substrate.
1.4. In Appendix 1, a plurality of optical scanning means respectively read sample spots on independent substrates.
[0116]
1.5. 2. The apparatus according to claim 1, further comprising a coarse positioning means for simultaneously changing positions of the plurality of optical scanning means on the sample surface.
1.6. Appendix 1, wherein the optical scanning means has a micromachine mirror.
1.7. In Appendix 1,
Having an optical fiber for guiding the light from the light source to the light collecting means,
It has a separation unit that separates the return light from the test section from the optical path from the light source, and detects the light separated by the separation unit with the light detection unit,
The optical fiber and the light collecting means are confocal or near confocal.
[0117]
1.7.1. In addition 1.7, the optical fiber which guided the light source light is branched by an optical coupler and guided to a plurality of optical scanning means.
1.8. Appendix 1 At
The apparatus has a plurality of wavelength selecting means for dividing return light from the subject by wavelength, and a plurality of optical scanning means.
1.8.1. Appendix 1.8, there is provided a reflected light detecting means for directly detecting a part of the return light.
[0118]
1.9. In Appendix 1,
This is a fluorescent probe in which a sample spot is arranged at a predetermined position on a substrate.
1.9.1. In Supplementary Note 1.9, the fluorescent probe is a DNA probe.
1.9.2. In Supplementary Note 1.9, the fluorescent probe is an antibody probe.
1.9.3. In Supplementary Note 1.9, the fluorescent probe is a protein probe.
1.10. In Supplementary Note 1, the sample spot is a tissue section arranged at a predetermined position on the substrate.
[0119]
1.11. In Supplementary Note 1, the sample spot is a set of cells arranged at a predetermined position on the substrate.
1.11.1. In Supplementary Note 1.11, there is a cell for arranging cells at a position defined on the base material.
[0120]
【The invention's effect】
As described above, according to the present invention, since the optical scanning means is provided with the automatic positioning means for performing the automatic positioning of at least one degree of freedom, the optical scanning means is automatically positioned in the optical axis direction of the condensing means or the like. By optically scanning the sample spot at high speed, it is possible to read out the fluorescence information from the sample spot while suppressing variations in detection characteristics.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of an optical scanning device according to a first embodiment of the present invention.
FIG. 2 is a longitudinal sectional view showing a configuration of an optical scanning optical system on the distal end side of the optical scanning probe.
FIG. 3 is a front view showing the configuration of the scanner in FIG. 2;
FIG. 4 is a view showing a coarse actuator and the like for positioning and holding a probe unit.
FIG. 5 is a diagram showing a configuration of a fine movement actuator provided near a base end of the optical scanning probe.
FIG. 6 is an explanatory diagram showing a state of a scanning method using sample spots arranged on a DNA array.
FIG. 7 is a flowchart showing an example of a process of positioning a three-dimensional position before actually performing optical scanning.
FIG. 8 is a view showing a state in which different optical arrays are scanned by different optical scanning probes.
FIG. 9 is a view showing a scanning method using sample spots arranged on a DNA array in the case of FIG. 8;
FIG. 10 is a perspective view showing a main part when optical scanning is performed by positioning and holding the optical scanning probe two-dimensionally.
FIG. 11 is a configuration diagram of a main part in an optical scanning device according to a second embodiment of the present invention.
FIG. 12 is a vertical cross-sectional view showing the configuration on the distal end side of the optical scanning probe.
FIG. 13 is a perspective view showing the optical scanning optical unit in FIG. 12;
FIG. 14 is a perspective view showing a peripheral portion of a probe unit according to a third embodiment of the present invention.
FIG. 15 is an explanatory diagram of a manufacturing method for preparing a large number of tissue specimen arrays.
FIG. 16 is a perspective view showing a cell culture container in which a number of cultured cell cells are arranged.
[Explanation of symbols]
1. Optical scanning device
2 ... Slide glass
3. Probe unit
3A-3C: Optical scanning probe
4 positioning means
5 XY scanning stage
6. XY scanning stage driving device
7A to 7C ... Detection unit
8 ... PC
9 Monitor
10. Data holding device
11a ... Ar laser
12a-12c ... Dichroic mirror
14a-14d: Optical fiber
17a ... Optical fiber
19 ... Optical scanning optical system
21 ... Tip
23 ... Scanner (scan mirror)
27 ... Focus
30 ... Scanner driving device
40 ... light detection section
42 ... Half mirror
43a-43c ... Dichroic mirror
44a-44c ... PMT
45a-45d ... A / D converter
46 Photodetector (PD)
51A ... fine movement actuator
52 Voice coil motor
55 ... DNA array
61, 62 ... bimorph piezoelectric element
63… Laminated piezoelectric element
65 ... Sample spot
57a-57c ... observation range

Claims (1)

An optical scanning device for detecting fluorescence from a plurality of sample spots, particularly microgene spots or microbio spots,
The sample spot is disposed on a substrate,
A light source,
Light-collecting means for condensing and emitting light from the light source to the test portion,
An optical scanning unit that two-dimensionally scans a focal point condensed on the part to be measured by the condensing unit in a direction orthogonal to an optical axis direction of the condensing unit;
Having a plurality of the light scanning means;
Having light detection means for detecting return light from the test portion,
An optical scanning device, comprising: an automatic positioning unit that performs automatic positioning of the optical scanning unit with at least one degree of freedom.

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