JP2003057180A - Fluorescent image measuring apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve a fluorescent image measuring apparatus that has an inexpensive configuration and can easily position a sample. SOLUTION: The fluorescent image measuring apparatus, in which a sample is irradiated with an excitation light which spreads two-dimensionally, and fluorescence generated from a fluorescent substance which has adhered to the sample is measured to detect the sample, is provided with a two-dimensional light receiving element for receiving excitation light that is transmitted through the sample or is reflected on the sample surface, and a movement means for moving the position of the sample based on an observation image by the light reception element.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
The present invention relates to a fluorescence image measuring device for measuring a biochip such as a protein, and particularly to positioning of a sample.

[0002]

2. Description of the Related Art Conventionally, a so-called fluorescence image measuring device for measuring a fluorescence image by using, for example, an epi-illumination type confocal laser microscope is well known. As the epi-illumination type confocal laser microscope, for example, JP-A-5-6 filed by the applicant of the present application
There is an "optical scanner for confocal point" described in No. 0980.

In order to position the sample with such an apparatus, a dedicated positioning means is required. Although such a positioning means is not shown in the above-mentioned confocal optical scanner, for example, "Application of confocal laser microscope to medicine and biology" published by Interdisciplinary Planning Co., Ltd. (March 28, 1995) This can be realized by applying a positioning method using transillumination as described on pages 10 to 11 of Japanese publication).

FIG. 4 is a specific example thereof. The laser light (excitation light) is focused by a plurality of microlenses 2 formed on the light-condensing disk 1 and focused on the pinhole of the pinhole disk 4. The condensing disc 1 and the pinhole disc 4 are arranged so that the pinholes are arranged at the focal positions of the plurality of microlenses.
Are connected through and rotate integrally.

The excitation light emitted from the pinhole is the lens 6
Then, the light becomes parallel light, and then the objective lens 8
Focus on top. When the fluorescent substance attached to the sample 9 is irradiated with the excitation light, fluorescence is generated. The fluorescent light passes through the objective lens 8 and the lens 6 and is condensed on the pinhole of the pinhole disk 4, and a fluorescent image of the sample surface is formed here.

The fluorescence passing through the pinhole is reflected by the dichroic mirror 3 arranged between the condensing disc 1 and the pinhole disc 4, passes through the lens 11 and the barrier filter 12, and forms an image on the light receiving surface of the camera 13. The barrier filter 12 is a filter that allows fluorescence to pass but removes background light of other wavelengths. The beam splitter 7
Are removed from the optical path during fluorescence measurement.

According to such a structure, it is possible to optically scan with the laser beam (multi-beam) and photograph the fluorescence image of the surface of the sample 9 with the camera 13. At this time, the sample 9 is observed prior to observation in the horizontal direction (direction perpendicular to the optical axis: hereinafter referred to as XY direction) and the vertical direction (optical axis direction: hereinafter referred to as Z).
Positioning (positioning) is performed as follows.

With the beam splitter 7 inserted in the optical path, a light spot is applied to the sample 9 from an illumination system arranged on the lower surface side of the sample 9, and the spot image on the upper surface of the sample 9 is visually confirmed through an observation system. As the illumination system, the light from the illumination light source 14 is collimated by the lens 15 as shown in the figure, and then the sample 9 is illuminated as Koehler illumination.

The light absorbed or scattered by the sample 9 is emitted from the sample surface to the upper side, enters the objective lens 8, is reflected by the beam splitter 7, and is then guided to the lens 10.
Thereby, the image on the sample can be visually observed through the lens 10.

When aligning the sample 9 in the XY directions,
The moving mechanism moves the sample 9 in the X and Y directions to position the observation portion of the sample. Since a well-known moving mechanism can be used, description and illustration of its configuration are omitted. Although there are many applications in which the movement of a specific protein in a cell is observed by fluorescence, in this case, the whole cell cannot be observed only by fluorescence. In this method, the entire cell can be observed in a transmission image, and therefore it is easy to move the cell to the center of the screen.

When the sample 9 is to be aligned in the Z direction, the sample 9 is moved in the Z direction (the moving mechanism is not shown) to align the observed image with the sharpest position.

[0012]

However, such a positioning mechanism is not only poor in operability but also large in size and expensive, and in addition, it takes time and effort to remove and insert the beam splitter from the optical path. There was a problem.

An object of the present invention is to solve the above problems, and to realize a fluorescence image measuring device having a simple and inexpensive structure and capable of easily positioning a sample.

[0014]

In order to achieve such an object, the invention of claim 1 irradiates excitation light to a two-dimensionally spread sample and measures fluorescence generated from a fluorescent substance attached to the sample. In a fluorescence image measuring device configured to detect a sample by doing so, a two-dimensional light receiving element that receives the excitation light that has passed through the sample or reflected on the sample surface, and the position of the sample based on the observation image by this light receiving element It is characterized by comprising a moving means for moving the.

According to this structure, the sample can be positioned by skillfully utilizing the excitation light which has not been used conventionally and which has passed through the sample or reflected on the sample surface.

In this case, if the sample can be moved in the optical axis direction by using the autofocus mechanism, the positioning of the sample in the optical axis direction can be automated and the operability is good. Become.

Further, the excitation light for irradiating the sample is the excitation light multi-beamed by the microlens. And irradiation with multi-beam,
Either optical scanning with a confocal optical scanner as in claim 4 or non-scanning type in which a sample is simultaneously irradiated with multiple beams as in claim 5 may be employed. When scanning with multiple beams, there is also an advantage that speckles do not occur in the observation image of the camera even if laser light is used as the light source.

[0018]

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in detail below with reference to the drawings. FIG. 1 is a block diagram showing an embodiment of a fluorescence image measuring device according to the present invention. In the figure, the same parts as those of FIG. 1 is different from FIG. 4 in that an illumination system portion including an illumination light source 14 and lenses 15 and 16 and a beam splitter 7 are provided.
The light spot observation system portion consisting of the lens 10 and the lens 10 is removed, and a lens 21 and a camera 22 having a two-dimensional light receiving element are used in place of it.

The lens 21 focuses the excitation light transmitted through the sample 9 on the light receiving surface of the camera 22. As a result, the two-dimensionally spread samples are respectively irradiated with the multi-beams of the excitation light, and the sample images can be observed by the camera 22. In this case, it is possible to observe the whole image of the sample 9 which is not a slice.

Since the sample 9 is scanned by the multi-beam, speckle does not occur in the observation image of the camera 22 even if the laser is used as the light source. The conventional confocal fluorescence microscope does not use the excitation light transmitted through the sample 9, but the present invention utilizes this to align the sample 9, which is a feature of the present invention.

The alignment of the sample 9 in the XY directions is performed by checking the observation image from the camera 22. Further, the alignment in the Z direction can be performed by an autofocus mechanism (not shown). The movement in the XYZ directions is not limited to the sample, and the excitation light side may be moved by moving the objective lens 8 in the XYZ directions.

As the autofocus mechanism, for example, in the case of the maximum contrast method, the Z of the sample is adjusted so that the amplitude of light and dark of the image in the observation image of the camera 22 becomes maximum.
A mechanism for automatically controlling the movement in the direction can be applied.

FIG. 2 shows another embodiment of the present invention. Figure 1
2 is an optical scanning type (scan type) confocal microscope, whereas FIG. 2 shows a case of a non-optical scanning type (scanless type) microscope. 2, parts that are the same as those in FIG. 1 are given the same reference numerals, and descriptions thereof will be omitted.

In FIG. 2, reference numeral 21 is a light collecting substrate in which a plurality of microlenses 22 are arranged on a transparent substrate. 23
Is a sample, and for example, a DNA chip or a DNA microarray in which samples are arranged two-dimensionally can be applied. In this case, the microlens 22 and the sample 2
The three sites are arranged in a one-to-one correspondence with each other.

In such a structure, the light collecting substrate 21
Laser light (excitation light) incident from above is condensed by each microlens 22, and each site of the sample 23 is irradiated with this condensed beam. After that, as in the case of FIG. 1, the fluorescence emitted from the sample 23 is reflected by the dichroic mirror 3, and the lens 1
The light is incident on the light source 1, is condensed, passes through the barrier filter 12, and forms an image on the light receiving element of the camera 13.

On the other hand, the excitation light transmitted through the sample 23 is condensed by the lens 24 on the light receiving element surface of the camera 25. The sample 23 is aligned based on the observed image of the light receiving element surface. The alignment of the sample is the same as in the case of FIG. 1, and is performed while checking the observation image of the camera 25 in the XY directions, and the position is automatically adjusted in the Z direction by an autofocus mechanism that operates based on the observation image. It is determined.

In such a scanless type fluorescence microscope, it is necessary that the beam and the site have the same positional relationship, and the alignment by such a configuration in FIG. 2 is extremely useful. It should be noted that an XYZ positioning marker may be prepared on the sample 23 side and alignment in the XYZ directions may be performed using this marker as a reference.

FIG. 3 is a diagram showing still another embodiment according to the present invention. 2 shows a scanless reflection type fluorescence microscope, while FIG. 3 shows a scanless transmission type fluorescence microscope. In the figure, the same parts as those in FIG. 2 are designated by the same reference numerals.

The fluorescence generated in sample 23 is
The light passes through the lens 3 and enters the lens 31 to become parallel light.
Incident on 2. On the way, light having a wavelength other than fluorescence (referred to as background light) is removed by the barrier filter 12 inserted between the lenses 31 and 32. The fluorescence from which the background light has been removed is condensed by the lens 32 and forms an image on the light receiving element surface of the camera 13.

The reflected light (excitation light) from the sample 23, which is used for aligning the sample, is reflected by the beam splitter 7.
Is reflected by the lens 11 and is incident on the lens 11 and is focused by the lens 11 and condensed on the light receiving element surface of the camera 13.

According to such a configuration, the alignment of the sample can be performed in the same manner as in the case of FIG. 2 based on the observation image of the sample surface formed on the light receiving element.

[0032]

As described above, the present invention has the following effects. (1) The position of the sample can be easily aligned by using the excitation light that has not been used conventionally and that has been transmitted through the sample or the excitation light reflected by the sample surface. (2) The configuration for aligning the sample is simpler and cheaper than the conventional one, and the fluorescence image measuring device excellent in operability can be easily realized. (3) The present invention can be applied to any of a scanning type or a scanless type, and also a transmission type or a reflection type fluorescent microscope, and the effect thereof is great in practical use.

[Brief description of drawings]

FIG. 1 is a configuration diagram showing an embodiment of a fluorescence image measuring device according to the present invention.

FIG. 2 is a configuration diagram showing another embodiment of the present invention.

FIG. 3 is a configuration diagram showing still another embodiment of the present invention.

FIG. 4 is a configuration diagram showing an example of a fluorescence image measuring device that can be realized by combining conventional devices.

[Explanation of symbols]

1 Focusing disc 2,22 micro lens 3 dichroic mirror 4 pinhole disc 5 drums 6,11,24,31,32 lens 7 Beam splitter 8 Objective lens 9,23 samples 12 Barrier filter 13,22,25 camera 21 Light collecting substrate

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) H04N 1/04 H04N 1/04 106Z 5C072 106 EF term (reference) 2G043 AA03 BA16 CA09 EA01 FA01 FA02 GA07 GB01 HA01 HA09 JA02 KA02 KA09 LA03 2H052 AA07 AA08 AA09 AC04 AC05 AC15 AC27 AC34 5B047 AA15 AA17 BB04 BC05 BC11 BC16 CA12 CB23 5B057 AA10 BA02 CA12 CD02 DA07 5C051 DA20 DB02 DA30 DA02 DB02 DB30 DA02 DC30 DC03 DC02 DC01 DC02 DC02 DB30 DB03 DC07 DC02 DC01 DC02 DC07 DC01 HA02 HA06 HA10 RA04 VA01

Claims (6)

[Claims] 1. A fluorescence image measuring device configured to detect a sample by irradiating a two-dimensionally spread sample with excitation light and measuring fluorescence generated from a fluorescent substance adhering to the sample. A fluorescence image measuring device comprising: a two-dimensional light receiving element that receives the excitation light reflected on the surface; and a moving unit that moves the position of the sample based on an observation image by the light receiving element. 2. The fluorescence image measuring apparatus according to claim 1, wherein the moving means is configured to move the sample in the optical axis direction by an autofocus mechanism. 3. The fluorescence image measuring device according to claim 1, wherein the sample is irradiated with excitation light that is converted into multi-beams by a microlens. 4. The fluorescence image measuring device according to claim 3, wherein the sample is configured to be optically scanned by a multi-beam confocal optical scanner. 5. The sample is configured to be simultaneously illuminated by multiple beams.
The fluorescence image measurement device described. 6. The fluorescence image according to claim 1, wherein the sample is provided with a marker for positioning in the XYZ directions, and the alignment in the XYZ directions is performed with reference to the marker. Measuring device.