JP2002207009A - Scanning type fluorometric apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning type fluorometric apparatus capable of two- dimensionally scanning a specimen of a two-dimensionally wide range at a high speed and capable of being constituted inexpensively. SOLUTION: The scanning type fluorometric apparatus is equipped with the specimen (11) labelled with a fluorescent substance with exciting light (L3), a first scanning means (14) for performing the reciprocating scanning of the specimen by subjecting exciting light to reciprocating movement along one direction (x), a second scanning means (17y) for moving the first scanning means (14) along the direction (y) crossing one direction (x) and a light detection means (15) for detecting the fluorescence (L4) generated from the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning fluorescence measuring apparatus for measuring fluorescence by two-dimensionally scanning a sample labeled with a fluorescent substance with spot-like excitation light.

[0002]

2. Description of the Related Art Conventionally, a scanning fluorescence which scans a sample two-dimensionally using excitation light condensed in a spot shape and sequentially measures fluorescence emitted from the sample excited at each light condensing point. Measurement devices are known. In such an apparatus, when the sample is two-dimensionally scanned, the spot-like excitation light Ls is generally moved in the xy directions on the sample.

More specifically, in the conventional scanning fluorescence measuring apparatus, a galvano-reflective mirror (x) rotatable about an axis parallel to the y-direction to reciprocate the excitation light Ls in the x-direction.
A galvanometer (x) for reciprocatingly rotating the reflection mirror (x), and a galvanometer reflection mirror (y) rotatable around an axis parallel to the x direction to move the excitation light Ls in the y direction. A galvanometer (y) for rotating the reflection mirror (y).

The above-mentioned reflection mirrors (x) and (y) are arranged on the optical path of an optical system for guiding the excitation light Ls to the sample. For this reason, the fixed sample is two-dimensionally rotated by reciprocating the reflecting mirror (x) at high speed using the galvanometer (x) and rotating the reflecting mirror (y) at low speed using the galvanometer (y). Can be scanned. Since the conventional scanning fluorescence measurement apparatus mainly observes a specimen (sample) on a slide glass, its scanning area is about 20 mm × 20 m.
m and very narrow.

[0005]

However, in recent years,
A two-dimensionally wide sample (for example, 200 mm × 20) in which DNA is electrophoresed on a sheet-like membrane or gel
It is also desired to measure the fluorescence of a sheet-like sample having a thickness of about 0 mm using the excitation light condensed in a spot shape.

[0006] However, in the above-mentioned conventional scanning fluorescence measuring apparatus, when two-dimensional scanning of a sample is performed, two reflecting mirrors (x) and (y) and galvanometers (x) and (y) are used. Was difficult to scan two-dimensionally. In the above-mentioned scanning fluorescence measurement apparatus, since one scanning area is very narrow (about 20 mm × 20 mm), when the stage is moved for two-dimensional scanning of a wide range of the sample, the number of movements increases. However, there is a problem that processing time such as image processing becomes long.

[0007] Further, since two reflecting mirrors (x) and (y) and two galvanometers (x) and (y) are provided, there is a problem that the cost is increased. SUMMARY OF THE INVENTION It is an object of the present invention to provide a scanning fluorescence measurement apparatus which can two-dimensionally scan a wide range of samples at high speed and can be configured at low cost.

[0008]

According to the present invention, there is provided a scanning fluorescence measuring apparatus comprising: an irradiating means (13) for irradiating a sample (11) labeled with a fluorescent substance with excitation light (L3); First scanning means (14) for reciprocally scanning the sample by reciprocating on the sample in one direction (x), and first scanning means
(14) is moved along the direction (y) intersecting the one direction (x) by scanning the sample in the intersecting direction (y). fluorescence
(L4).

According to the scanning fluorescence measuring apparatus of the present invention described above, the excitation light (L3) is reciprocated in one direction (x) by the first scanning means (14), and the second scanning means (1).
Since the first scanning means (14) is moved in the intersecting direction (y) by 7y), a wide area of a large sample can be two-dimensionally scanned at high speed.

[0010]

Embodiments of the present invention will be described below in detail with reference to the drawings.

An embodiment of the present invention is defined as claims 1 to 4.
Corresponding to The scanning fluorescence measurement apparatus 10 of the present embodiment
As shown in FIG. 1, a sample stage 12 on which a sample 11 is placed,
Excitation light (L3) from below for the sample 11 on the sample stage 12
Irradiating section 13, a galvano mirror section 14 for periodically reciprocating the excitation light (L3) in the x direction, and a light receiving section 15 for receiving fluorescence (L4) generated from the sample 11.
And a part of the irradiation unit 13 described above, the galvanometer mirror unit 14,
An optical head 16 accommodating the light receiving unit 15 and the optical head 1
The stage 6 includes a stage 17 for moving the stage 6 in the xy directions, and a controller 18 for controlling the above-described units.

The sample table 12 of the scanning fluorescence measuring apparatus 10
Even if the sample 11 has a size of about 200 mm × 200 mm, it is a fixed base on which the sample 11 can be stably mounted, and is formed of a transparent plate member (for example, a glass plate). The mounting surface of the sample table 12 is parallel to the xy directions. The sample 11 placed on the sample stage 12 is, for example, DNA or protein dispersed in a membrane or gel by electrophoresis, and is labeled with a fluorescent substance. When the entire surface of a large sample 11 (for example, 200 mm × 200 mm) is to be measured, as shown in FIG. 2, the sample 11 is divided into a plurality of strip-shaped scanning areas 11a, 11b, 11c,.
Dimensionally scanned. The width Dx of the scanning areas 11a, 11b, 11c,... Is about 10 mm.

As will be described later in detail, in the scanning fluorescence measuring apparatus 10 of the present embodiment, the two-dimensional scanning of each scanning area 11a is performed by the galvanomirror unit 14 with the excitation light.
While reciprocating (L3) in the x direction, the stage unit 17
To move the excitation light (L3) in the y direction. Further, the movement to the next scanning area 11a is performed by moving the excitation light (L3) in the x direction by the stage unit 17.

Next, the scanning fluorescence measuring device 10 shown in FIG.
Each of the constituent elements (13 to 18) will be described in detail. The irradiation unit 13 (irradiation means) includes a laser light source 21, an optical fiber 22, a collimator lens 23, a filter 24,
And a condenser lens 25. Laser light source 21
Is a light source that continuously emits laser light, and is disposed outside the optical head 16. The optical fiber 22 guides laser light emitted from the laser light source 21 into the optical head 16.

The collimating lens 23 includes an optical fiber 22
Laser light L0 emitted from the end face of
This is an optical element that converts the optical axis into 1, and its optical axis is aligned in the z direction perpendicular to the xy directions (the mounting surface of the sample stage 12).

The filter 24 is an optical element that receives the parallel laser light L1 from the collimator lens 23 and transmits the laser light L2 in a specific wavelength band. The specific wavelength band corresponds to a wavelength band in which the fluorescent substance used for labeling the sample 11 can be excited. The condenser lens 25 includes the filter 2
An optical element (for example, a focal length f = 50 mm) for condensing the transmitted light (laser light L2) from the sample 4 on the sample 11 so that its optical axis is perpendicular to the xy directions (the mounting surface of the sample table 12). Placed in The laser light L3 condensed by the condensing lens 25 has a spot shape (having a diameter of about 10 μm).

The filter 24 of the illumination unit 13 described above
The dichroic mirror 3 of the light receiving unit 15 described later is located between the
5 and a reflection mirror 31 of the galvanometer mirror unit 14 described later
And are arranged. Therefore, the laser light L2 transmitted through the filter 24 is reflected by the dichroic mirror 35 and the reflection mirror 31, and then reaches the condenser lens 25.

Here, the optical path of the laser light L2 between the filter 24 and the dichroic mirror 35 is parallel to the z direction. The optical path (incident optical path) of the laser light L2 between the dichroic mirror 35 and the reflection mirror 31 is parallel to the x direction. The optical path (reflected optical path) of the laser beam L2 between the reflection mirror 31 and the condenser lens 25 is oblique with respect to the x direction and the z direction, and includes the optical axis of the condenser lens 25 and x It is included in a plane (paper surface) parallel to the direction. The optical path of the laser beam L3 between the condenser lens 25 and the sample 11 will be described later.

According to the irradiating section 13 described above, the sample 11 is irradiated with the laser beam L3 condensed by the condenser lens 25 as excitation light. When the fluorescent substance of the sample 11 is excited by the laser beam L3, the sample 11 emits fluorescence L4. Next, the galvanometer mirror unit 14 (first scanning unit) will be described. The galvanometer mirror unit 14 includes a reflection mirror 31 disposed on an optical path of the laser light L2,
And a galvanometer 32 (reciprocating drive unit).

The reflection mirror 31 is rotatable, and its rotation axis 31a is arranged in the y direction. When the reflection mirror 31 rotates, the direction of the reflection light path of the laser beam L2 changes according to the rotation angle of the reflection mirror 31. The reflection mirror 31 is arranged on the pupil of the condenser lens 25. Therefore, the optical path of the laser beam L3 between the condenser lens 25 and the sample 11 is always parallel to the optical axis of the condenser lens 25 regardless of the rotation angle of the reflection mirror 31. However, the position of the optical path of the laser light L3 changes according to the rotation angle of the reflection mirror 31.

The galvanometer 32 is a drive device for periodically rotating the reflection mirror 31 back and forth around a rotation axis 31a. The reciprocating rotation of the reflection mirror 31 by the galvanometer 32 is performed based on a control signal from the control device 18 (for example, 300 Hz). When the reflection mirror 31 is reciprocally rotated by the galvanometer 32, the laser light L
The reflected light path 2 and the optical path of the laser light L3 reciprocate periodically according to the rotation angle of the reflection mirror 31. As a result, the position where the laser beam L3 is incident on the sample 11 also periodically reciprocates along the x direction according to the rotation angle of the reflection mirror 31. Note that the laser beam L3 is perpendicularly incident on the sample 11.

Here, assuming that the range of the rotation angle of the reflecting mirror 31 is Δθ, the angle range in which the reflected light path of the laser beam L2 changes is 2 · Δθ, and the range Δx of the reciprocating movement of the laser beam L3 is (condensing lens 25 focal length f) × tan (2 · Δ
θ). By the way, the fluorescent light L4 emitted from the sample 11 by the irradiation of the laser light L3 (excitation light) is condensed by the condensing lens 25 of the irradiating section 13 and reflected by the reflecting mirror 31 of the galvano mirror section 14 to be laser light. The light is guided to the incident light path of L2.

In order to receive the fluorescent light L4 guided to the incident light path of the laser light L2 as described above, a light receiving section 15 is provided. The light receiving unit 15 includes a dichroic mirror 35, a filter 36, a condenser lens 37, and a photomultiplier tube (PMT) 38 disposed on the incident optical path of the laser beam L2.
And a signal processing unit 39.

The dichroic mirror 35 is an optical element that reflects the short-wave laser light L2 and transmits the long-wave fluorescence L4. According to the dichroic mirror 35, the extremely weak fluorescent light L4 guided to the incident light path of the laser light L2 can be separated from the incident light path of the laser light L2. The filter 36 is arranged on the optical path of the fluorescence L4 separated by the dichroic mirror 35. Filter 36
Is an optical member that transmits fluorescent light L4 of a specific wavelength.
According to the filter 36, even if the laser light L2 becomes stray light and is guided to the light receiving section 15, it can be completely removed.

The condenser lens 37 is an optical element for condensing the fluorescent light L4 on the light receiving surface of the photomultiplier tube 38. The photomultiplier tube 38 photoelectrically converts and amplifies the fluorescent light L4 condensed on the light receiving surface, and outputs an electric signal proportional to the fluorescent light amount to the signal processing unit 39. The signal processing unit 39 discretely takes in the electric signal output from the photomultiplier tube 38 based on the control signal from the control device 18. An image processing device (image processing means) (not shown) is connected to the signal processing unit 39, and a display device (not shown) is connected to the image processing device.

The signal processing unit 39 A / D converts the discretely captured electric signals, stores the converted signals in a memory,
(Not shown) and output to a display device (not shown). As a result, the display device displays the fluorescence L4 emitted from the sample 11.
Is displayed in a pseudo color. Now, the optical head 16 houses therein the laser light source 21 of the irradiation unit 13, the galvanometer mirror unit 14, and the light receiving unit 15.

The stage section 17 for moving the optical head 16 is composed of a stage 17x (moving means) for moving the optical head 16 in the x direction and a stage 17y (second scanning means) for moving the optical head 16 in the y direction. Have been. The movement of the stages 17x and 17y is performed based on a control signal from the control device 18. Control device 18 (control means)
The signal processing unit 39 and the stage 1 correspond to a reference signal (for example, 300 Hz) when controlling the galvanometer 32.
7x, 17y (for example, a computer). The control device 18, the galvanometer 32, and the signal processing unit 39 are connected via a cable (not shown).
Further, a monitor and an input device (not shown) are connected to the control device 18.

Further, information on the size of the sample 11, information on a plurality of measurement areas in the sample 11, and the like are prepared in advance in the memory of the control device 18. Information on the plurality of measurement areas is appropriately displayed on a monitor (measurement area setting screen). Next, the operation of fluorescence measurement in the scanning fluorescence measurement device 10 configured as described above will be described. FIG. 3 shows a scanning fluorescence measuring device 10.
4 is an operation flowchart of FIG.

In step S1, the control device 18
A measurement area setting screen is displayed on the monitor, and one or more measurement areas (scanning areas 11a and 11a in FIG. 2) to be measured are displayed.
1b,...) From the measurement area setting screen. Note that the measurement area may be selected by the user inputting numerical values of two xy coordinates on two diagonal lines of the measurement area (rectangular shape) on the measurement area setting screen on the monitor.

Thereafter, in step S2, the controller 18 scans the laser beam L3 at high speed in the x direction (high-speed reciprocating scan) and moves the optical head 16 in the y direction to acquire a partial image of the sample 11. . The partial image of the sample 11 is an image of one of the one or more measurement areas selected in step S1 (for example, the scanning area 11a in FIG. 2).

As described above, when the galvanometer 32 reciprocates the reflection mirror 31 based on the control signal from the control device 18 (for example, 300 Hz), the incident position of the laser beam L3 on the sample 11 is set along the x direction. Reciprocate periodically. For example, when the angle range Δθ of the reciprocating rotation of the reflection mirror 31 is 5.7 °, the range Δx of the reciprocating movement of the laser beam L3 is about 10 mm (when the focal length f of the condenser lens 25 is 50 mm).

In parallel with the reciprocation, the control device 18
The optical head 1 is controlled by controlling the stage 17y.
6 is linearly moved in the y direction at a constant speed. The movement of the optical head 16 at this time is slower than the above-described reciprocating movement. By moving the optical head 16 in the y direction,
The laser beam L3 also linearly moves at a constant speed in the y direction.

As described above, the laser beam L3 is reflected by the reflection mirror 3
1. By moving the stage 17y at a constant speed in the y direction while reciprocating in the x direction by the galvanometer 32 (step S2), the incident position of the laser beam L3 on the sample 11 is in a zigzag shape shown in FIG. The orbit 10a will be drawn. As a result, of sample 11,
One scanning area 11a shown in FIG. 2 is two-dimensionally scanned by the spot-like laser light L3.

Then, the fluorescent light L4 emitted from the scanning area 11a of the sample 11 by the irradiation of the laser light L3 enters the photomultiplier tube 38 (FIG. 1) and is converted into an electric signal.
The signal is taken into the signal processing unit 39 at regular intervals at discrete timings based on a control signal from the control device 18.
As a result, the image processing apparatus connected to the signal processing unit 39
Image data (fluorescence amount) corresponding to each xy coordinate of the fluorescence image of the scanning area 11a is transferred to (not shown) (step S3). A display device connected to the image processing device;
(Not shown) includes the fluorescence L from the scanning area 11a of the sample 11.
4 is displayed.

Here, when using a laser beam L3 having a spot diameter of 10 μm to capture a fluorescent image of the scanning area 11a (FIG. 2) of the sample 11 at a resolution of 10 μm, the laser beam L3
Is the spot diameter (10 μm) along the track 10a (FIG. 4).
It is preferable that the fluorescent light L4 from the scanning area 11a of the sample 11 be taken into the signal processing unit 39 every time the movement is performed.

Further, the laser beam L3 (spot diameter 10 μm)
m), the sample 11 is two-dimensionally scanned by using both the forward path and the return path of the reciprocating movement (300 Hz), and the fluorescent image of the scanning area 11a (FIG. 2) of the sample 11 is captured at a resolution of 10 μm. Laser light L3 (FIG. 1)
Is about 6 mm / sec.

When the two-dimensional scanning of one scanning area 11a of the sample 11 reaches the final end in the y direction, the controller 18 moves the optical head 16 in the y direction to terminate the two-dimensional scanning of the scanning area 11a. To stop. At this time, a fluorescent image synthesized based on the image data of the scanning area 11a is displayed on a display device (not shown) (step S4).

When the two-dimensional scanning of one scanning area 11a of the sample 11 has been completed, the laser beam L3 has moved by 200 mm in the y direction, so if the scanning speed in the y direction is 6 mm / sec, one The time required for two-dimensional scanning of the scanning area 11a is about 33.3 seconds. Next, the control device 18 determines in step S5
All the measurement areas selected in step S1 (FIG. 2
It is determined whether the fluorescence measurement has been completed for the scan areas 11a, 11b,...), And if an unmeasured measurement area remains, the stage 17x is controlled to control the optical head 16 (that is, the laser beam L3). Is moved in the x direction (step S6).

For example, when the scanning area 11b (FIG. 2) located next to the scanning area 11a is to be measured next, the movement of the optical head 16 (laser light L3) in the x direction is caused by the width Dx ( 10 mm). The width Dx of the scanning area 11a corresponds to the distance of the reciprocating scanning by the laser light L3.

The control device 18 moves the optical head 16 in the y direction by controlling the stage 17y (step S7). At this time, the movement of the optical head 16 in the y direction is opposite to the distance from the last end to the start end of the two-dimensional scanning (the y-direction of the scanning areas 11a, 11b,...) In the two-dimensional scanning (step S2). Length) minutes.
Thereby, the position of the optical head 16 in the y direction is returned to the original position.

Thereafter, the control unit 18 returns to step S2, and at a constant speed (6 mm / sec) in the y direction while reciprocating the laser beam L3 in the x direction, similarly to the two-dimensional scanning of the scanning area 11a described above. Move. At this time, the incident position of the laser beam L3 on the sample 11 draws a zigzag orbit 10b similar to the orbit 10a shown in FIG. As a result, the scanning area 11b adjacent to the scanning area 11a shown in FIG. 2 is two-dimensionally scanned with the spot-shaped laser light L3.

The fluorescent light L4 emitted from the scanning area 11b of the sample 11 by the irradiation of the laser light L3 is taken into the signal processing section 39 at regular intervals, as in the case of the above-described scanning area 11a. Fluorescence L from
The image data (fluorescence amount) corresponding to each xy coordinate of the fluorescent image based on No. 4 is transferred to an image processing device (not shown) (step S3). The display device (not shown) includes a scanning area 1.
1b is displayed.

The controller 18 controls the scanning area 11 of the sample 11
When the two-dimensional scan for b reaches the final end in the y direction, the movement of the optical head 16 in the y direction is stopped in order to end the two-dimensional scan for the scan area 11b. At this point, a fluorescent image synthesized based on the image data of the scanning area 11b is displayed on a display device (not shown) (step S4).

Next, in step S5, the control device 18 determines whether or not the fluorescence measurement has been completed for all the measurement areas (scanning areas 11a, 11b,... In FIG. 2) selected in step S1. If the measurement area for the measurement remains, the processes in steps S6 and S7 are performed, and then the process returns to step S2. As described above, the control device 18 of the scanning-type fluorescence measurement device 10 causes the stage 17y to move at a constant speed in the y-direction while the laser beam L3 is reciprocated in the x-direction by the galvanometer mirror unit 14 (step S2). And the optical head 16
The moving step (step S6) of moving the (laser light L3) by a fixed amount (the width Dx of the scanning area 11a) in the x direction by the stage 17x is alternately and repeatedly executed.

Then, the control device 18 executes the above step S
All the measurement areas selected in 1 (scanning area 11 in FIG. 2)
When the fluorescence measurement for “a, 11b,...” is completed (Step S5 becomes Y), the image data acquired for all the measurement areas is stored, and then the fluorescence measurement operation is completed (Step S8). Therefore, according to the scanning fluorescence measurement apparatus 10 of the present embodiment, even when the entire surface of the large sample 11 (for example, 200 m × 200 mm) is the measurement target,
The sample 11 is divided into a large number of scanning areas 11a, 11b,... (FIG. 2), and the scanning areas 11a, 11b,. Is obtained.

When the size of each of the scanning areas 11a, 11b,... Is 10 mm × 200 mm, the sample 11 (200 mx
200mm)
The above-described scanning step is performed 20 times. If the time required for one scanning step is about 33.3 seconds,
The time required to obtain a fluorescent image of the entire surface of the sample 11 (200 m × 200 mm) is about 666 seconds.

In the above-described scanning fluorescence measuring apparatus 10, the laser beam L3 is reciprocated at a high speed in the x direction (for example, 300 Hz) by the reciprocating rotation of the reflection mirror 31, and the optical head 16 (laser beam L3) is moved by the stage 17y.
Are moved in the y direction, the scanning areas 11a, 11b,... Of the large sample 11 can be two-dimensionally scanned at high speed. Further, in the above-described scanning fluorescence measuring apparatus 10, the moving step is performed each time one scanning step is completed, and the moving step is performed in the previous scanning step.
Since another area (scanning area 11b) adjacent to the area that has been dimensionally scanned (for example, the scanning area 11a) is to be scanned in the next scanning step, these scanning steps and the moving step are alternately repeated. The entire large sample 11 can be two-dimensionally scanned at high speed.

The scanning fluorescence measuring apparatus 10 of the present embodiment
This is particularly effective when a large area of a large sample 11 (200 m × 200 mm) is to be roughly investigated with a low resolution (for example, 10 μm). Further, in the above-described scanning fluorescence measurement apparatus 10, since only one galvanometer mirror section 14 is provided, the cost of the apparatus can be reduced.

Further, since the reflecting mirror 31 of the galvanometer mirror section 14 can be arranged on the pupil of the objective lens (condensing lens 25), illumination unevenness can be reduced. Further, in the above-described scanning fluorescence measuring apparatus 10, the laser light source 21 is disposed outside the optical head 16, and the laser light from the laser light source 21 is guided into the optical head 16 by the optical fiber 22. The weight and size of the optical head 16 can be reduced.

In the above-mentioned scanning fluorescence measuring apparatus 10, the fixed large sample 11 (for example, 200 m × 20
0 mm), the optical head 16 is moved in the xy direction, so that the size of the entire apparatus is reduced as compared with a configuration in which a large sample 11 is moved in the xy direction as in a scanning fluorescence measurement device 50 (FIG. 7) described later. Can be achieved. In the above-described embodiment, an example has been described in which the fluorescence L4 condensed by the condenser lens 37 is guided to the photomultiplier tube 38. However, as in the case of the scanning fluorescence measurement device 40 shown in FIG. A pinhole 41 may be arranged at the focal position of 37, and a photomultiplier tube 38 may be arranged after the pinhole 41. By guiding the fluorescence L5 after passing through the pinhole 41 to the photomultiplier tube 38, unnecessary light (noise component) in the depth direction of the sample 11 can be removed. As a result, a fluorescent image with good contrast can be obtained.

In the above embodiment, the laser light source 21 is disposed outside the optical head 16.
6 may be arranged inside. Furthermore, in the above-described embodiment, the photomultiplier tube 38 is disposed inside the optical head 16, but may be disposed outside the optical head 16. In this case, the end face of the optical fiber is arranged at the focal position of the condensing lens 37, and the fluorescent light is applied to the optical head 16 by the optical fiber.
May be guided to the photomultiplier tube 38 arranged outside the box. By disposing the photomultiplier tube 38 outside the optical head 16,
The weight and size of the optical head 16 can be reduced.

In the above-described embodiment, an example in which the sample 11 is fixed and the optical head 16 is moved in the xy directions has been described. However, as in the case of the scanning fluorescence measuring device 50 shown in FIG. The sample 11 may be fixed and moved in the xy directions. In the scanning fluorescence measurement apparatus 50, the stage 17 (FIGS. 1 and 4) for moving the optical head 16 is omitted, and instead, the stage (51x, 51y) for moving the sample 11 in the xy directions. ) Is provided. The stage section (51x, 51y) converts the sample 11 to x
It comprises a stage 51x for moving in the direction and a stage 51y for moving in the y direction.

The laser head 21 is built in the optical head 16 of the scanning fluorescence measuring apparatus 50. Also,
A reflection mirror 52 is arranged between the collimator lens 23 and the dichroic mirror 35 in order to reduce the height of the optical head 16 and stabilize it. In the scanning fluorescence measurement apparatus 50 shown in FIG. 7, the laser beam L3 is reciprocated at high speed in the x direction (for example, 300 Hz) by the reciprocating rotation of the reflection mirror 31, and the sample 11 is moved in the y direction by the stage 51y. .. (FIG. 2) of the sample 11 can be two-dimensionally scanned at high speed.

In the scanning fluorescence measuring device 50, when the two-dimensional scanning (scanning step) for one scanning area 11a is completed, the stage 51x is controlled to control the sample 1
1 is moved in the x direction (moving step). The amount of movement of the sample 11 at this time corresponds to the width Dx of the scanning areas 11a, 11b,... (FIG. 2). Thus, the scanning fluorescence measuring device 5
0, the moving step is performed each time one scanning step is completed, and another area (the scanning area 11a) adjacent to the area (for example, the scanning area 11a) two-dimensionally scanned in the previous scanning step.
Since b) is to be scanned in the next scanning step, by repeating these scanning step and moving step alternately, the entire large sample 11 can be two-dimensionally scanned at high speed. This scanning fluorescence measuring device 50 also has a large sample 11 (200 m × 20
This is particularly effective when a wide range (0 mm) is roughly investigated with a low resolution (for example, 10 μm).

Incidentally, an LED may be used instead of the laser light source 21. Further, a photodiode may be used instead of the photomultiplier tube 38.

[0056]

As described above, according to the present invention,
A large area (or the whole) of a large sample can be two-dimensionally scanned at high speed. Further, an inexpensive device can be provided.

[Brief description of the drawings]

FIG. 1 is an overall configuration diagram of a scanning fluorescence measurement device 10 of the present embodiment.

FIG. 2 shows a large sample 11 having a large number of scanning areas 11a, 11
It is the figure which showed a mode that it divided | segmented into b, ....

FIG. 3 is an operation flowchart of the scanning fluorescence measurement apparatus 10.

FIG. 4 is a diagram for explaining the operation of fluorescence measurement in the scanning fluorescence measurement apparatus 10.

FIG. 5 is a diagram illustrating an operation of fluorescence measurement in the scanning fluorescence measurement apparatus 10.

FIG. 6 is an overall configuration diagram of a scanning fluorescence measurement device 40 of a modified example.

FIG. 7 is an overall configuration diagram of a scanning fluorescence measurement device 50 of a modified example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 Scanning fluorescence measuring apparatus 11 Sample 12 Sample stand 13 Irradiation part 14 Galvano mirror part 15 Light receiving part 16 Optical head 17 Stage part 18 Control device

Claims (4)

[Claims] An irradiating means for irradiating a sample labeled with a fluorescent substance with excitation light; and a reciprocating scan of the sample by reciprocating the excitation light in one direction on the sample. A first scanning unit, a second scanning unit that scans the sample in the intersecting direction by moving the first scanning unit in a direction intersecting the one direction, A scanning fluorescence measurement device comprising: a light receiving unit that receives fluorescence. 2. The scanning fluorescence measurement apparatus according to claim 1, further comprising a sample stage on which the sample is fixedly mounted, wherein the first scanning unit is arranged on an optical path of the irradiation unit. Having a reflection mirror, and having a reciprocating drive unit for reciprocatingly rotating the reflection mirror about a predetermined axis, by periodically reciprocating the excitation light along the one direction perpendicular to the axis. Means for reciprocatingly scanning the sample, wherein the second scanning means scans the sample placed on the sample table by moving the first scanning means along the intersecting direction. A scanning fluorescence measuring device, characterized in that it is a means. 3. The scanning fluorescence measurement apparatus according to claim 1, wherein the first scanning unit is moved by a predetermined amount in the one direction, and Control means for setting the constant amount according to the width of reciprocal scanning, and alternately controlling two-dimensional scanning of the sample by the first scanning means and the second scanning means and movement by the movement means; An image processing means for forming a two-dimensional image based on an image signal of the sample obtained by the light receiving means as a result of the scanning control by the control means. . 4. An irradiating means for irradiating a sample labeled with a fluorescent substance with excitation light, and a reflecting mirror arranged on an optical path of said irradiating means, wherein said reflecting mirror reciprocates around a predetermined axis. By having a reciprocating drive to rotate, by periodically reciprocating the excitation light along one direction perpendicular to the axis,
A first scanning unit that reciprocally scans the sample, by moving at least one of the first scanning unit and the sample along a direction that intersects the one direction, thereby moving the sample in the intersecting direction. A second scanning unit that scans, a light receiving unit that receives fluorescence generated from the sample, a moving unit that moves at least one of the first scanning unit and the sample by a certain amount in the one direction, The constant amount is set according to the width of the reciprocal scanning by the first scanning means, and the two-dimensional scanning of the sample by the first scanning means and the second scanning means and the movement by the moving means are performed. Control means for alternately controlling; and image processing means for forming a two-dimensional image based on an image signal of the sample obtained by the light receiving means as a result of scanning control by the control means. Scanning fluorescence measuring apparatus characterized by a.