JP6661947B2 - Fluorescent image capturing apparatus and fluorescent image capturing method - Google Patents

Description

The present invention relates to fluorescent imaging instrumentation 置及 beauty fluorescent image capturing method for capturing a fluorescence produced in the specimen.

  A microchamber is known as an apparatus for efficiently analyzing a chemical reaction of a sample within a short time. In the microchamber, the sample does not emit light due to a chemical reaction, and emits fluorescence when exposed to excitation light. In the analysis using a microchamber, a sample in an active state is observed using fluorescence or luminescence as a clue using a device such as an optical microscope or a digital microscope capable of enlarging the sample. The sample is a biomolecule such as a protein or a nucleic acid, and information relating to the function and activity of the biomolecule can be obtained by observation.

In the above-described observation of biomolecules, measuring the characteristics of biomolecules one by one in an individually identified state is referred to as “single-molecule measurement”. In some cases, up to about three are unavoidably included. In single-molecule measurement, biomolecules can be observed by labeling with a fluorescent dye, even though the resolution of an optical microscope is on the order of the wavelength of visible light of several hundred nm. In single-molecule measurement, it is necessary to keep the biomolecules in the sample active.
Handling samples in aqueous solution. The aqueous solution holding the sample is hereinafter referred to as “sample solution”.
A micro-chamber sealed with a sample containing a biomolecule held therein is referred to as a “biomolecule analysis chip” in this specification. The single-molecule measurement using the fluorescence microscope of the biomolecule analysis chip does not determine the intensity of the fluorescence generated by the sample, but determines the fluorescence by using two values, “present” and “absent”. For this reason, observation of a biomolecule analysis chip does not require a dedicated large-scale fluorescence microscope capable of linearly quantifying the fluorescence value, but includes an arithmetic unit (Central Processing Unit) and a digital camera. It is considered that fluorescence can be detected even by using a portable communication device such as a smart phone.

  The biomolecule analysis chip basically has a configuration like a slide glass and a cover glass, like the slide. Many small holes are regularly formed in the slide glass or the cover glass, and the sample solution is sandwiched between the slide glass and the cover glass. The sample solution fills the inside of the microcontainer, and there is 0 to 1 biomolecule inside each hole. Occasionally, there may be up to about three.

In order to identify the biomolecule to be observed, a fluorescent label is added to each of the biomolecules. Also, in order to easily observe the biomolecules, colloidal gold or fine particles are added to the biomolecules. As the fine particles, polystyrene beads, magnetic beads, and the like are used. By adding fluorescent labels and microparticles to biomolecules, it is possible to visualize molecules of a size that are difficult to see with the resolution of an optical microscope in single-molecule measurement. However, in such an observation method, the object to be observed is a single molecule or a few molecules of a biomolecule, and it is necessary that the objects are dispersed without being aggregated so that each of them can be identified. The condition is that the concentration of the target substance (protein) in the sample solution is extremely low as compared with a normal biochemical experiment (for example, about 10 pM (picomolar: about 10 ⁻⁹ mol / L)). .

In biochemical, chemical, or medical experiments, microfluidic systems in which a chemical analysis system is miniaturized using micromachine technology (MEMS: micro electro mechanical system) are used. In a microfluidic system, a microchamber having a fine channel structure is formed using an etching technique, a photolithography technique, or the like. The microchamber of the microfluidic system is used as a chemical reaction container for causing various biochemical or chemical reactions on a small volume of fluid.
Hard materials such as silicon and glass are used as materials for the microchamber of the microfluidic system, and polymers such as polydimethylsiloxane (PDMS), aliphatic cyclic polyolefin (COP) and its copolymer (COC) are used. A soft substance such as resin or silicone rubber is used.

  The reaction time in the case of movement only by diffusion of biomolecules is generally represented by a value obtained by dividing the square of the distance between particles to be reacted by the diffusion coefficient. Specifically, when the diffusion movement distance is 1 μm, it takes 0.1 second, and when the distance is 1 cm, it takes about 10 million seconds. The reaction efficiency of biomolecules will increase when the particles are in a range that can move with each other in seconds. The range that can be moved in a few seconds is 10 μm or less. For this reason, in a microchamber of a microfluidic system, a microchannel having a moving distance of 10 μm or less is used.

In such a microfluidic system, a photodetector is used to detect the fluorescence of biomolecules in the macro chamber. Photodetectors for detecting fluorescence are described in Patent Literature 1 and Non-Patent Literature 1. Patent Literature 1 describes that an object to be imaged is irradiated with LED light, photographed by a commercially available digital camera, and analyzed. Patent Literature 1 does not describe how to focus on photographing an object.
Non-Patent Document 1 describes that a fluorescent image of a microparticle or a virus is captured by a smartphone.

JP, 2011-521237, A

A. ozcan et.al, ACS Nano, 2013, 7 (10), pp 9147-9155, Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone.

However, in the microchamber, the volume of the hole is as small as several to several tens of fL, and the number of biomolecules in the inside is small, so that the fluorescence emitted from the biomolecules is very weak. The sensitivity of the digital camera can be detected even if the fluorescence is weak even if it is accumulated for several seconds. However, if autofocus cannot be detected in real time, the sensitivity of the autofocus sensor is insufficient and it is difficult to focus on the fluorescent light emitting surface. If the light emitting surface is out of focus, the captured fluorescence may be darker than it actually is in the image and may not be visible on the image.
In autofocusing, the position of a lens at which the contrast or the luminance is maximized is searched for in real time and focused. The biomolecule analysis chip has a laminated structure of air, glass, air or liquid, glass, and air from the lower layer. When such a biomolecule analysis chip is irradiated with auxiliary light for focusing, the focus position is aligned with the uppermost glass surface having the strongest reflection, and the focus is adjusted to the desired back surface of the uppermost glass or to the desired position. Cannot fit the surface of the second glass.

Furthermore, in order to image a biomolecule in a microchamber, a lens is used in a macro mode so that a pattern having a size of several μm is resolved. For this reason, the depth of focus of the lens becomes 100 μm or less, and it is difficult to detect a very dark fluorescent light which is blurred even if integration is performed when focusing is not performed with high accuracy.
Some digital cameras, such as single-lens reflex cameras, have autofocus and manual focus functions.However, such cameras are more expensive than digital cameras that have only the autofocus function, which is easily mass-produced and inexpensive. is there.

  In addition, digital cameras built into smartphones must be small, and most of them are 10 mm or less in length and width. For this reason, in a small digital camera with a built-in smartphone, it is difficult to provide a mechanism for manually sending out the lens and adjusting the focus in terms of space. It is also conceivable to operate the lens with a program, but for a smartphone manufacturer, the camera control program is a core technology, and the program is usually not disclosed. For this reason, many cameras provided in smartphones adopt autofocus customized by each camera manufacturer. Even if it becomes slightly larger, it would be more desirable if manual focus could be performed in addition to autofocus.

As described above, in order to photograph the fluorescence of a sample in a biomolecule analysis chip using a digital camera, it is necessary to focus on a desired position while using an autofocus mechanism. If the sample surface can be photographed in a focused state, the obtained plurality of images can be subjected to image processing to reduce noise and detect dark fluorescence.
The present invention has been made in view of the above problems, and an object thereof is to provide a fluorescent image photographing instrumentation 置及 beauty fluorescence imaging method that can be focused at any position of the biomolecule analysis chip .

  According to one embodiment of the present invention, there is provided a fluorescence image capturing apparatus in which a mounting portion for mounting a molecular analysis chip holding a sample solution including a fluorescent member and an imaging device having an autofocus function are fixed. A fixed part, a distance adjusting part for adjusting a relative distance of the mounting part with respect to the fixing part, and the fluorescent member on the molecular analysis chip on the mounting part whose distance is adjusted by the distance adjusting part. And a light irradiator for irradiating the exciting light.

  The fluorescence image capturing method according to one embodiment of the present invention is a fluorescence capturing method for capturing fluorescence generated in a molecular analysis chip having a plurality of layers, wherein an optical element is provided in one of the layers of the molecular analysis chip. Irradiating the emitted light, focusing the camera on the layer irradiated with the light, fixing the focus by using an autofocus function of the camera, and the molecular analysis chip And moving the camera to an arbitrary position on the molecular analysis chip.

The molecular analysis chip according to one embodiment of the present invention includes a transparent first layer, a transparent second layer provided so as to overlap the first layer, and holding a sample solution together with the first layer; A first edge light corresponding to the first layer and including an optical member and a resin member having light guiding property; and a second edge light corresponding to the second layer and including an optical member and a resin member having light guiding property. It is desirable to have an edge light.

Fluorescence imaging instrumentation 置及 beauty fluorescence imaging method that can be focused at any position of the biomolecule analysis chip can be provided.

It is a figure for explaining an embodiment of a fluorescence image photography device. FIG. 2 is a diagram for explaining the inside of the fluorescence image capturing apparatus shown in FIG. 1. It is the figure which illustrated the fluorescence image which was image | photographed. It is a figure for explaining embodiment of a biomolecule analysis chip. FIG. 5 is a diagram for explaining a microwell of the biomolecule analysis chip shown in FIG. 4. It is a figure for explaining LED edge light of a biomolecule analysis chip. FIG. 3 is a diagram showing a state in which a biomolecule analysis chip is set on a sample holder. It is a figure for explaining an embodiment of a fluorescence image photography method.

Hereinafter, a fluorescence image capturing apparatus according to an embodiment of the present invention will be described.
(Constitution)
FIGS. 1A and 1B are perspective views for explaining a fluorescent image capturing apparatus 1 according to the present embodiment. FIGS. 1A and 1B illustrate the fluorescent image capturing apparatus 1 from different angles. Is shown.
The fluorescence image capturing apparatus 1 of the present embodiment includes a mounting section 35 on which the biomolecule analysis chip 30 holding a sample solution including a fluorescent member is mounted, a fixing section 51a for fixing a smartphone 51 having an autofocus function, The Z-axis stage feed knob 208, which is a distance adjusting unit that adjusts the relative distance of the mounting unit 35 to the fixed unit 51a, and the mounting unit 35 whose height has been adjusted by the Z-axis stage feed knob 208. A semiconductor laser 56 which is a light irradiator for irradiating the biomolecule analysis chip 30 with light for exciting the fluorescent member.

In the above configuration, the Z-axis stage feed knob 208 adjusts the relative distance of the mounting section 35 to the fixed section 51a, and thereby the camera of the smartphone 51 fixed to the fixed section 51a and the living body on the mounting section 35. The distance from the molecular analysis chip 30 is adjusted.
Further, the fluorescence image capturing apparatus 1 holds an angle adjusting unit 202 for adjusting the angle of the semiconductor laser 56, a micrometer head 210 used for adjusting the angle by the angle adjusting unit 202, and a Z-axis stage feed knob 208. The semiconductor laser 56 includes a clamp 209 for fixing the semiconductor laser 56, a battery 206 for supplying power to the semiconductor laser 56, and a laser battery connection unit 203 for connecting the semiconductor laser 56 and the battery 206.

The battery 206 includes a battery power button 204 for instructing power supply by the battery 206 and a battery charging port 205 into which a cable for charging the battery 206 is inserted. The fixing portion 51a may have any configuration as long as it fixes the smartphone 51 to the sample holder 207.
Although not shown in FIG. 1, the biomolecule analysis chip 30 is provided with an LED edge light. The LED edge light will be described later in detail.
In the present embodiment as described above, members necessary for capturing a fluorescent image can be compactly integrated. In the present embodiment, a fluorescent image can be captured using a smartphone, which is a general-purpose portable communication terminal. For this reason, the fluorescence image capturing apparatus 1 of the present embodiment can realize a portable configuration.

Since the semiconductor laser 56 operates at about 5 V in the fluorescence image capturing apparatus 1, the semiconductor laser 56 that irradiates the laser beam for excitation can be operated by the battery 206 that is a USB power supply composed of a rechargeable lithium battery. it can. In the present embodiment, as the small-sized semiconductor laser 56 having low power consumption, a small-sized semiconductor laser which has a small heat generation and can be used for natural cooling of an aluminum block of 2 cm 3 or less is selected.
Such a semiconductor laser 56 has the advantage that it is inexpensive, but the wavelength of the laser light oscillated shifts in the long wavelength direction because the resonator expands with heat as the temperature rises. Further, in such a semiconductor laser 56, the wiring resistance increases due to the influence of heat, and the effective voltage is insufficient, so that the laser light output decreases. Such a characteristic is not preferable for measuring an analog fluorescence value because the absolute value of the data changes. However, in the present embodiment in which the fluorescence value is evaluated using two digital values, the fluorescence value does not change near the threshold value. As long as it does not matter. The laser light source of the device that measures the fluorescence value as an analog value must be kept at a constant temperature while being cooled by a Peltier element or the like. Not suitable for equipment.

When irradiating the sample with a biomolecule analysis chip with laser light, the angle and height of the semiconductor laser 56 can be adjusted. The angle of the semiconductor laser 56 is adjusted by an operator using the angle adjusting unit 202. The operator adjusts the height of the mounting section 35 using the Z-axis stage feed knob 208. With such a mechanism, the laser beam of the semiconductor laser 56 is obliquely applied to the biomolecule analysis chip 30. The fluorescence image capturing apparatus 1 of the present embodiment can optimally adjust the position and angle of the semiconductor laser 56 according to the shape and configuration of the biomolecule analysis chip 30.
The above configuration was selected as a result of exploring an optical system that is advantageous for thinning and compactness without using a fluorescent cube that minimizes background stray light that can completely separate excitation light and fluorescent light, such as a fluorescence microscope. It is a thing.

  The biomolecule analysis chip 30 has a sample solution sealed between two types of layers having different refractive indexes. The configuration in which the laser beam of the present embodiment is obliquely applied to the biomolecule analysis chip 30 has a characteristic that light obliquely incident on a layer is totally reflected at an interface of the layer at a specific incident angle. This is the focus. In the present embodiment, the angle adjusting unit 202 is provided to separate laser light and fluorescence used for exciting the fluorescent dye and to set an incident angle at which the background of the excitation light does not increase during fluorescence observation. The height of the semiconductor laser 56 with respect to the biomolecule analysis chip 30 can be adjusted by adjusting the position of the mounting portion 35 with the Z-axis stage feed knob 208.

FIG. 2 is a schematic diagram for explaining the inside of the fluorescence image capturing apparatus 1 shown in FIG. The configuration shown in FIG. 2 includes a smartphone 51 having a main body 55 and a camera 52, an objective lens 54 that is a condensing lens for condensing light generated by the excited fluorescent member, and a preset among the condensed light. And a fluorescence interference filter 53 that is an optical filter that selectively transmits light of the specified wavelength. Note that the camera 52 has an autofocus function.
The fluorescence interference filter 53 slides out when the observer pulls the ring 221 shown in FIG. With such a configuration, the fluorescence image capturing apparatus 1 can use an appropriate fluorescence interference filter 53 according to the fluorescence to be observed.

  The biomolecule analysis chip 30 is fixed at a position substantially at the focal length of the objective lens 54. The biomolecule analysis chip 30 has a substrate 32 and a cover glass 34 as shown in the figure, and the substrate 32 has a surface facing the cover glass 34 of the substrate 32 (hereinafter referred to as “front surface”) and a back surface (hereinafter referred to as “front surface”). The microwell 31 is formed as a plurality of fine holes that are recessed toward the “rear surface”. The microwell 31 has, for example, a circular shape with an opening of 5 μm in diameter. The diameter, depth, and arrangement of the microwell can be determined as appropriate.

The semiconductor laser 56 irradiates the laser light f obliquely to the microwell 31 from the back surface of the substrate 32. The laser light f travels from the biomolecule analysis chip 30 to the objective lens 54 as substantially parallel light. For this reason, the laser beam f incident on the fluorescence interference filter 53 becomes substantially parallel light, and the fluorescence interference filter 53 can exhibit the performance as specified. The fluorescence interference filter 53 transmits the fluorescence to be observed and can reduce the intensity of light outside the observation target to 1 / 100,000 or less. That is, the fluorescent dye is excited by the laser light f. The excited fluorescent dye emits fluorescence and stabilizes. At this time, in the fluorescence image capturing apparatus of the present embodiment, the selection ratio of the fluorescence to the laser light f can be set to 1:10 ⁷ or more.
Further, in the present embodiment, the laser light f is irradiated obliquely toward the back surface of the substrate 32. Since the laser light f is totally reflected on the surface of the substrate 32, the laser light f does not directly enter the fluorescence interference filter 53, and stray light can be reduced to 1 / 100,000 or less.

The camera 52 photographs the light transmitted through the fluorescence interference filter 53. FIG. 3 is a diagram illustrating a fluorescent image captured by the camera 52 as an example. As described above, in the present embodiment, the selectivity ratio of the fluorescent light to the laser light f is 1:10 ⁷ or more, and the stray light is 1 / 100,000. Therefore, only the fluorescent light 41 appears in the captured fluorescent light image. I have.
In the present embodiment, a camera built into the smartphone 51 is used as the camera 52. However, the present embodiment is not limited to the use of the camera of the smartphone 51, and any camera having an autofocus function is used to photograph the biomolecule analysis chip 30. There may be. It is more desirable to have a manual focus function in addition to the auto focus. Alternatively, the fluorescence interference filter 53 may be slid and removed, and replaced with another fluorescence interference filter. This makes it possible to use an interference filter for fluorescence that is optimal for the fluorescence used for processing the biomolecules.

(Biomolecule analysis chip)
4A and 4B are views for explaining the biomolecule analysis chip 30 of the present embodiment. FIG. 4A is a top view of the biomolecule analysis chip 30, and FIG. It is sectional drawing which follows the line segment 4b-4b.
As described above, the biomolecule analysis chip 30 has the substrate 32 and the cover glass 34. The substrate 32 has a microwell 31 formed therein. Further, a spacer 33 is inserted between the substrate 32 and the cover glass 34. The spacer 33 regulates the distance between the substrate 32 and the cover glass and adheres the two.
Note that the present embodiment is not limited to a configuration in which a sample solution contains a biomolecule, and can be applied to a molecular analysis chip for analyzing any molecule.

<Substrate>
The substrate 32 is a substrate having optical transparency. Permeability refers to the property of transmitting the fluorescence generated from the biomolecule encapsulated in the microwell 31 or its surroundings.
Fluorescence used for labeling a known biological sample often has an emission wavelength peak in a wavelength range of 400 nm to 700 nm, which is a visible light region. The transparent substrate 32 may be, for example, glass or resin. When the substrate 32 is a resin substrate, for example, acrylonitrile, butadiene, styrene copolymer synthetic resin (ABS resin), polycarbonate, cycloolefin copolymer (COC), acryl, polyvinyl chloride (PVC), polystyrene, polyethylene , Polyvinyl acetate, PET (polyethylene terephthalate), PEN (polyethylene naphthalate) and the like.

Further, in order to observe the fluorescence of the biomolecules in the microwell 31 from the back surface of the substrate 32, in addition to having transparency, the substrate 32 does not emit autofluorescence, or even if it emits, the autofluorescence is weak. It is preferable that the material does not hinder the fluorescence observation. A fused synthetic quartz glass is a material that has transparency and is so weak and negligible that auto-fluorescence does not hinder fluorescence observation of an observation target. Fused synthetic quartz glass is a fused quartz glass obtained by purifying impurities contained in natural quartz by melt synthesis.Structural defects due to impurities are removed, and auto-fluorescence mainly caused by emission centers due to structural defects can be reduced. it can. When auto-fluorescence occurs in fused synthetic quartz glass, the auto-fluorescence is very weak.
In addition, as a material that causes a difference between the fluorescence to be observed and the self-emission and does not hinder the fluorescence observation, there are low-fluorescence glass, acrylic resin, cycloolefin copolymer (COC), and the like.

  The thickness of the substrate 32 is preferably 1 mm or less, more preferably 0.17 mm, in order to observe fluorescence from the back surface of the substrate 32 with an optical microscope. By setting the thickness of the substrate 32 in such a range, the fluorescence generated in the biomolecule analysis chip 30 can be observed with the biological objective lens. In many cases, the thickness of the substrate 32 is in the range of about 0.5 mm to about 0.7 mm without any problem, and can be appropriately determined according to the situation.

On the surface of the substrate 32, there is a microwell layer 36 in which the microwell 31 is formed. The microwell layer 36 is hydrophobic, and the surface of the substrate 32 on the bottom surface of the microwell is hydrophilic. Therefore, the side surface of the microwell 31 is hydrophobic, and the surface is composed of hydrophilicity and hydrophobicity. I have. The microwell layer 36 is made of, for example, a resin. As the hydrophobic resin, for example, a photosensitive resin or a thermoplastic resin can be used. Like the material of the substrate 32, the photosensitive resin or the thermoplastic resin preferably emits no auto-fluorescence, or is weak even if it emits, and does not interfere with the fluorescence observation.
If the negative photosensitive resin is not inactivated (the structure is not significantly changed by light and does not react to light) after the shape is formed, the photo-crosslinking material may emit autofluorescence. Inactivate. In the case where a photosensitive resin is used, when there is a possibility that the film shape may "drip" due to the heat treatment, the exposure and development conditions are determined in anticipation of "whore" so that the film has a desired shape after the heat treatment.

The material of the hydrophobic layer constituting the microwell layer 36 has a contact angle with water of 80 ° or more, preferably 100 ° or more. Furthermore, the hydrophobic layer also needs to be compatible with oil, and is required to satisfy the required performance for both water and oil. For this reason, the material of the hydrophobic layer is not limited to the above numerical values, but is appropriately determined by a combination with another material.
Specifically, for example, the hydrophobic layer forming the microwell layer can be formed by selecting a material having low autofluorescence from a novolak resin of a photosensitive resin. Since the photosensitive resin has a functional group that reacts to light, select a material whose auto-fluorescence is weak and confirm that it does not interfere with normal fluorescence observation, and use it as the hydrophobic layer of the micro-chamber layer. It becomes possible.

<Microwell>
FIGS. 5A and 5B are diagrams for explaining the microwell 31. FIG. FIG. 5A is an image obtained by photographing the microwell 31 obliquely from the surface side of the substrate 32. FIG. 5B is an enlarged view of one of the microwells 31 shown in FIG. 5A. The substrate on which a plurality of microwells 31 are formed as shown in FIG. 5A is a microchamber in the sense of a container for microwells.
As shown in FIGS. 5A and 5B, in the present embodiment, the shape of the microwell is hereinafter referred to as the opening surface of the microwell as the upper surface, the bottom surface of the hole as the bottom surface, and the inner peripheral surface as the side surface. It shall be represented by the formed solid.

The microwell 31 has a shape and dimensions in which one microwell 31 accommodates only one or several biomolecules. The plurality of microwells 31 are arranged vertically and horizontally at the same interval, and a predetermined number of microwells 31 can be divided into groups at even larger intervals. The number of microwells in one divided group is not limited, but may be, for example, in the range of 100 to 10,000.
The total number of the microwells 31 formed on the substrate 32 may be, for example, 100,000 to 10,000,000, but can be appropriately determined depending on the type of the sample solution to be measured. Although the total number of microwells 31 is not limited, for example, when measuring the mutation of cell-free DNA, from the viewpoint that the mutation rate to be detected is 0.01%, for example, from 1 million to 2 million per cm ² It is preferable to arrange them in a range.

The microwell 31 is not limited to the prism shape shown in FIGS. 5 (a) and 5 (b). For example, it may be formed in a cylindrical shape. Moreover, even if it is a prism, the number of corners of the upper surface and the bottom surface may be further smaller (for example, a rectangular parallelepiped, a hexagonal prism, an octagonal prism, and the like). Further, the shape of the microwell 31 is not limited to a shape having the same shape and size as the top surface and the bottom surface, and may be a truncated cone or a truncated pyramid. Further, the bottom of the microwell 31 may be a curved surface (a convex surface or a concave surface).
The shape and dimensions of the microwell 31 are one per microwell 31 in consideration of the amount of a sample solution containing a biomolecule to be accommodated in the microwell 31 and the size of the beads to which the biomolecule is attached, or It is determined appropriately so as to accommodate several biomolecules.

  When the microwell 31 has a cylindrical shape, its size is, for example, a circle having a diameter of 3 μm to 50 μm on the top and bottom surfaces. When a sample solution containing biomolecules is sealed, the diameter of the top and bottom surfaces is preferably 3 μm to 3 μm. 10 μm. The depth of the microwell 31 is, for example, 3 μm to 50 μm, and preferably 3 μm to 5 μm. However, as described above, the dimensions of the microwell 31 are determined in consideration of the amount of the sample solution containing the biomolecules contained in the microwells 31 and the preferable ratio of the size of the beads to which the biomolecules are attached to the dimensions of the microwells 31. Is determined as appropriate.

<Sample solution>
The fluorescence image photographing apparatus of the present embodiment can observe the fluorescence emitted from the cells accommodated in the microchamber from the back surface side of the substrate 32 using an optical microscope. Before the biomolecule is housed in the microchamber, the biomolecule may be fluorescently labeled by treating it with a fluorescent dye capable of specifically labeling the biomolecule to be observed. In this embodiment, the biomolecules can be supplemented by using beads that specifically recognize the biomolecules. In such a method, the beads are subsequently housed in a microchamber and contacted with a fluorescent dye capable of specifically labeling the biomolecule, and the biomolecule is fluorescently labeled in the microwell.
The biomolecule treated as described above generates fluorescence in the microwell 31. The observer can observe the fluorescence from the back side of the substrate using, for example, an inverted microscope. By observation, the number of biomolecules to be observed can be specified.

  The encapsulation of the sample solution containing the biomolecule in the microwell is performed by filling the sample solution into the microwell and, after the sample solution is introduced into the well, sending oil to enclose the sample solution in the well. Process. The hydrophobic layer of the microwell layer preferably has a contact angle with water of 80 ° or more. Further, the contact angle of the hydrophobic layer of the microwell layer with the oil to be further enclosed is preferably 10 ° or less, and particularly preferably 7 ° or less.

<LED edge light>
FIG. 6 is a diagram for explaining the LED edge lights 11a and 11b provided corresponding to the cover glass 34 and the substrate 32 of the biomolecule analysis chip 30, respectively. FIG. 6A is a top view of the biomolecule analysis chip 30. FIG. 6B is a sectional view taken along line 6b-6b shown in FIG. 6A.
The LED edge light 11a shown in FIG. 6 includes an LED chip 12a, a prism 13a as an optical member, and an acrylic block 14a as a resin member. Specifically, for example, a prism 13a made of acrylic resin or optical glass such as BK7 is adhered to a 0.8 mm × 1.6 mm LED chip 12a. The prism 13a bends the direction of the light emitted by the LED chip 12a by 90 degrees. Further, the acrylic block 14 a serving as a light guide plate is in close contact with the cover glass 34 of the biomolecule analysis chip 30. The size of the prism 13a is 2 mm or less on one side. The LED edge light 11b has the same configuration as the LED edge light 11a, and the acrylic block 14b is in close contact with the substrate 32.

  The edge of the acrylic block 14a can be prevented from being damaged by sandwiching a resin having a thickness of several tens of μm and having good optical transparency on the contact surface between the cover glass 34 and the acrylic block 14a. Further, when the substrate 32 of the biomolecule analysis chip is made of glass, it is possible to prevent the edge of the acrylic block 14b from being damaged by sandwiching a resin having good optical transparency having a thickness of several tens of μm on the contact surface with the acrylic block 14b. it can. Here, as the resin sandwiched between the glass and the acrylic blocks 14a and 14b, a cyclic olefin polymer (COP) or a cyclic olefin copolymer (COC) is suitable.

FIG. 7A shows a state where the biomolecule analysis chip 30 is set on the sample holder 207. FIG. 7B shows the LED edge light 11a shown in FIG. As shown in FIG. 7A, a battery 206 is connected to the LED edge light 11a via a dip switch 15a. A battery 206 is connected to the LED edge light 11b via a dip switch 15b. The LED edge light 11a is turned on and off by turning on and off the dip switch 15a. The LED edge light 11b is turned on and off by turning on and off the dip switch 15b.
The LED edge lights 11a and 11b of the present embodiment are formed by connecting a resin optical fiber (for example, with a black resin cover having a diameter of about 0.5 mm) to an ultra-small resin molded LED and further forming an acrylic block 14a. , 14b may be bonded.

The light L emitted from the LED chip 12a is refracted at an angle of 90 degrees in the prism 13a as shown in FIG. The refracted light travels from the acrylic block 14a to the micro chamber.
As shown in FIG. 7, the position where the LED edge light 11a contacts the cover glass 34 and the position where the LED edge light 11b contacts the substrate 32 are shifted from each other in a top view. The contact position does not need to be the same, and is preferably different depending on layers such as a cover glass and a substrate, since light can be independently applied to each layer.

In the present embodiment shown in FIGS. 6 and 7, the direction of the LED edge light 11a with respect to the cover glass 34 and the direction of the LED edge light 11b with respect to the substrate 32 are the same. However, the present embodiment is not limited to such a configuration, and a configuration in which the directions of the LED edge lights 11a and 11b are different from each other is also preferable.
The sample holder 207 is designed so that light from the LED chip 12a is incident only on the cover glass 34 of the biomolecule analysis chip 30 and light from the LED chip 12b is incident only on the substrate 32. By setting the biomolecule analysis chip 30 in such a sample holder 207, it is possible to align the LED edge lights 11a and 11b with the cover glass 34 and the substrate 32.

<Fluorescence image capturing method>
FIG. 8 is a diagram for explaining a method of capturing a fluorescent image performed by the fluorescent image capturing apparatus of the present embodiment. Note that the procedure shown in FIG. 8 is manually performed by a photographer using a fluorescent image photographing apparatus.
In the present embodiment, first, in step S1, the LED edge light corresponding to the substrate or cover glass of the biomolecule analysis chip to be observed is turned on, and the autofocus function of the camera is used to focus on the surface of the cover glass.

After focusing in step S1, the photographer fixes the focus of the camera in step S2. Then, the lit LED edge light is turned off. In step S3, the photographer photographs the surface of the cover glass while irradiating the LED analysis chip with the laser light of the semiconductor laser.
By taking an image according to the above-described procedure, it is conceivable that even a dark image with low fluorescence intensity can be acquired by image processing later. Specifically, according to the present embodiment, it is possible to capture a plurality of focused images and perform image processing to extract an image buried in randomly generated noise such as thermal noise.

The fluorescent image capturing apparatus, the molecular analysis chip, and the fluorescent image capturing method of the present embodiment described above can focus on an arbitrary position of the molecular analysis chip.
In particular, in this embodiment, an LED edge light is provided on the end surface of the biomolecule analysis chip, and the substrate 32 and the cover glass 34 are independently irradiated with LED light. For this reason, each layer of the substrate 32 and the cover glass 34 can be focused by autofocus. In particular, in the biomolecule analysis chip used in the present invention, the surface of the substrate 32 that generates fluorescence is the same as the surface on which the microwell that shines by the LED light emitted from the end face (side) of the substrate 32 is the same. It is now possible to focus.
Further, in the present embodiment, the relative distance of the camera 52 fixed to the sample holder 207 with respect to the biomolecule analysis chip 30 can be changed by using the Z-axis stage feed knob 208, so that the focus can be finely adjusted. It is possible to adjust the focus and to surely focus.

(First embodiment)
The inventor of the present invention has produced a fluorescent image capturing apparatus in which the sample holder shown in FIG. 1 and a smartphone are combined. In the fluorescent image capturing apparatus, a 0.8 mm × 1.6 mm white LED edge light manufactured by Nichia Corporation (manufacturer) and a custom-made plastic prism were used as the LED edge light. Such an LED edge light irradiates LED light by selecting a cover glass and a substrate constituting a biomolecule analysis chip, respectively. Then, the cover glass and the substrate were separately photographed according to the procedure shown in FIG.
Microwells having a diameter of about 5 μm are regularly arranged on the surface of the substrate. The surface of the substrate is illuminated by an LED edge light, and the surface is set to a fixed focus using the software of a smartphone camera. After that, the LED edge light was turned off, and the laser light was irradiated obliquely from behind the substrate, so that an in-focus image with dark fluorescence was captured. FIG. 3 described above is an image captured by the above procedure.

(Second embodiment)
The second embodiment is the same as the first embodiment until the LED edge light is turned on, the camera is focused on the cover glass or the substrate, and fixed. Next, in the second embodiment, the relative distance of the camera to the biomolecule analysis chip is changed using the Z-axis stage feed knob, and the camera is focused on the surface of the cover glass or the substrate at a plurality of distances. In the second embodiment, the cover glass and the substrate surface can be photographed at a plurality of arbitrary focus positions.
When the magnification is large, the distance between the objective lens and the subject becomes short. For this reason, the focus may be deviated between the central portion and the outer peripheral portion of the lens. In the second embodiment, an image focused on concentric circles can be obtained by photographing images at a plurality of focus positions.

  The fluorescence image capturing apparatus of the present invention described above can be used in a medical compact device or a bedside of a hospital or the like. For example, the fluorescence modification of a small volume, that is, a picoliter or less volume used for detection of a single molecule biological substance can be used. A fluorescence image of the array of the sample solution or the sample droplet thus obtained can be taken.

DESCRIPTION OF SYMBOLS 1 Fluorescence image imaging device 11a, 11b Edge light 12a, 12b LED chip 13a, 13b Prism 14a, 14b Acrylic block 15a, 15b Dip switch 30 Biomolecular analysis chip 31 Microwell 32 Substrate 33 Spacer 34 Cover glass 35 Mounting part 36 Micro Well layer 41 Fluorescence 51 Smartphone 51a Fixed part 52 Camera (Smartphone built-in)
53 Fluorescence interference filter 54 Objective lens 56 Semiconductor laser 202 Angle adjustment unit 203 Laser battery connection unit 204 Battery power button 205 Battery charging port 206 Battery 207 Sample holder 208 Z-axis stage feed knob 209 Clamp (for fixing Z-axis stage)
210 Micrometer head 221 ring (handle for extracting sample holder)
310 faces

Claims (6)

A mounting portion for mounting a molecular analysis chip holding a sample solution containing a fluorescent member,
A fixing unit for fixing a photographing device having an autofocus function,
A distance adjustment unit that adjusts a relative distance of the placement unit with respect to the fixing unit,
A light irradiation unit that irradiates the molecular analysis chip on the placement unit with the distance adjusted by the distance adjustment unit with light that excites the fluorescent member;
An angle adjustment unit that sets an incident angle of the light irradiated by the light irradiation unit,
Equipped with a,
The fluorescence image capturing apparatus, wherein the light irradiating unit irradiates the light from a diagonal direction to a light irradiating surface of the molecular analysis chip, which is a surface opposite to the surface on the imaging device side .   A condenser lens for condensing light generated by the fluorescent member irradiated with light by the light irradiator, and an optical filter for selectively transmitting light of a preset wavelength among the condensed light The fluorescence image capturing apparatus according to claim 1, comprising:   The fluorescence image capturing apparatus according to claim 2, wherein the optical filter is replaceable. The molecular analysis chip is a cover glass located on the imaging device side, a substrate located on the opposite side to the imaging device side, and located between the cover glass and the substrate, and can accommodate biomolecules. A microwell layer having at least one microwell,
The light from the light irradiating unit is irradiated obliquely toward the microwell from the side of the substrate opposite to the imaging device side, according to any one of claims 1 to 3, wherein The fluorescent image capturing apparatus according to the above. A fluorescence imaging method for imaging fluorescence generated in a molecular analysis chip having a plurality of layers,
Irradiating one of the layers of the molecular analysis chip with light emitted by an optical element,
Focusing a camera on the layer irradiated with the light,
Fixing the focus using an autofocus function of the camera;
On the light irradiation surface, which is the surface opposite to the camera-side surface of the molecular analysis chip, the light for generating the fluorescent light is totally reflected at the interface between the layers constituting the molecular analysis chip. Irradiating the light irradiation surface from an oblique direction,
Moving the camera relative to the molecular analysis chip and focusing the camera at an arbitrary position on the molecular analysis chip. After fixing the focus, further comprising a step of turning off the optical element,
6. The fluorescence image capturing method according to claim 5, wherein after the optical element is turned off, light for generating the fluorescence is irradiated.