JP4883398B2 - Background light reduction method and member for evanescent wave excitation fluorescence observation - Google Patents

Description

  The present invention relates to a method for reducing background fluorescence in evanescent wave excitation fluorescence observation and a member used in the method.

  The fluorescence observation method based on the evanescent wave excitation method is a technology that has been highly successful in recent years, such as enabling single-molecule observation of proteins in the field of microscopy.

  The fluorescence observation method using such an evanescent wave excitation method is based on the evanescent wave generated in a limited number of hundreds of nanometers in the vicinity of the interface when the light is totally reflected in the substrate in contact with the fluorescently labeled probe solution on the substrate. This is based on the selective excitation of fluorescently labeled probe molecules in the vicinity of the substrate-liquid layer interface. That is, incident light is incident from the end face of the slab type waveguide substrate, and is repeatedly reflected and guided through the slide substrate to generate an evanescent wave, and the fluorescence emitted by the probe molecules excited by the evanescent wave is observed. Thus, the interaction between the molecule immobilized on the surface of the slide substrate and the probe molecule can be observed (FIG. 1).

  Such fluorescence observation by evanescent wave excitation is particularly suitable for observing the binding state between the above-mentioned molecule and a fluorescent probe in a microarray in which a large number of test molecules are immobilized. Microarray technology using such evanescent wave excitation method For example, by injecting a fluorescent probe sample solution into a reaction vessel installed on a substrate having a slab waveguide installed in a horizontal direction, the fluorescent probe sample is brought into contact with the slab waveguide. In some cases, by introducing incident light at an angle in the waveguide, it is possible to selectively excite the probe molecules in the vicinity of the interface by the evanescent wave generated on the waveguide (patented) In addition, the inventor has filed several applications (PCT-JP2004009600, PCT-JP2004019333, Japanese Patent Application 2005-184171).

  However, the selective excitation of the evanescent wave excitation method is theoretically a phenomenon that depends on the function of the distance from the interface, not the selectivity for the interface-bound molecule, and completely eliminates the excitation of non-bonded molecules existing far above the interface. It's not going to be zero. In reality, there is stray light that deviates from the total reflection condition that occurs non-ideally in the device, and it is known that a phenomenon in which the probe solution layer above the evanescent region shines is observed in actual observation. It was. Such stray light or light that does not satisfy the total reflection condition while traveling in the waveguide reaches the upper layer of the probe solution and directly excites the probe molecules, resulting in increased background light intensity, resulting in a large S / N ratio. Cause a significant decline. This has made it difficult to achieve both a sufficient S / N ratio and sensitivity with the conventional evanescent excitation method. In addition, when the background light is strong, the use of the high-concentration probe solution becomes a limiting factor, which makes it difficult to detect weak interactions.

  In contrast, the present inventors, as a countermeasure against stray light entering the reaction vessel on the substrate in the evanescent wave excitation fluorescence detection, disperse the colloid by adding a colloid solution to the probe liquid layer, and the probe solution by stray light. A method has been developed to make it difficult for the generated fluorescence to reach the detector even when the upper layer is excited (Japanese Patent Application 2005-006298). Although this method can easily attenuate background light, the equilibrium reaction may change depending on the contents of the added colloid solution, and the probe molecules adsorb to the colloidal particles. In some cases, it is not sufficient because it is unsuitable.

Further, the present inventors have taken a step further as a countermeasure against stray light entering the reaction tank on the substrate in evanescent wave excitation fluorescence detection, by making the probe solution layer extremely thin, stray light inside the reaction tank. A technology has been developed to reduce the absolute number of fluorescent molecules in the upper layer of the probe solution that is excited when passing through (Japanese Patent Application No. 2006-203257). However, none of these techniques is a method of dealing with the source of stray light itself that excites the fluorescent molecules in the upper layer of the probe solution, because it is a method of suppressing the influence of stray light existing, There was a natural limit to the lower limit of background light reduction. There has been no report on the fundamental countermeasure technology for removing the presence of stray light from the reaction tank.

Special table 2003-521684 gazette

The problem of the present invention is that when performing evanescent wave excitation fluorescence observation using a substrate having a slab waveguide, stray light generated under optically non-ideal conditions is generated in an evanescent field in a reaction vessel on the substrate. It is a method of achieving fluorescence observation at a high S / N ratio by effectively suppressing the phenomenon of reaching the upper part, and the suppression of the stray light can be carried out easily and inexpensively. Another object of the present invention is to provide a new means for suppressing stray light, which can be applied to a fully automated analyzer.

  The present inventor has found that non-ideal stray light remaining in the evanescent wave excitation fluorescence observation apparatus deviates from the optical design intention, or the inside of the slab type waveguide substrate while being totally reflected by being incident inside the waveguide. Focusing on the fact that stray light that deviates from the total reflection condition reaches the upper layer of the fluorescent probe solution on the substrate and excites fluorescent molecules, this is a major cause of background light. By effectively arranging the stray light absorption region on the substrate as a means to suppress reaching the bath, the amount of stray light reaching the upper layer of the probe solution layer is suppressed, and the background during evanescent wave excitation fluorescence observation A significant reduction in light was achieved and the present invention was completed.

That is, the present invention is as follows.
(1) After introducing a probe solution containing a fluorescently labeled probe molecule into a reaction vessel having a waveguide substrate having a test molecule immobilized on the bottom, the immobilized molecule and the probe molecule are bonded, and then the waveguide substrate. When performing evanescent wave excitation fluorescence observation by introducing light into the reaction vessel, it is possible to reduce the background light in the evanescent wave excitation fluorescence observation by suppressing the incidence of stray light into the reaction tank, from the incident light incident end. The background light reduction method as described above, wherein a stray light absorption region having a width of at least 3 mm or more in the direction of incident light is provided on the upper surface or the lower surface of the waveguide substrate while reaching the test molecule fixing position.
(2) The background light reducing method as described in (1) above, wherein the stray light absorption regions are provided on both the upper and lower surfaces across the waveguide substrate.
(3) The background light reduction method according to (1) or (2) above, wherein the stray light absorption region is formed by adhesion of a light absorption member.
(4) The background light reducing method according to any one of (1) to (3) above, wherein the incident light incident end face of the waveguide substrate is laser-cut.
(5) The background light reduction method according to any one of (1) to (4), wherein the reaction tank is formed of a light absorbing member.
(6) A waveguide substrate used for evanescent wave excitation fluorescence observation, which has a reaction vessel in which a test molecule is fixed at the bottom, and the top surface of the waveguide substrate between the incident light incident end and the test molecule fixing position or The waveguide substrate according to claim 1, wherein a stray light absorption region having a width of at least 3 mm or more in the incident light direction is provided on the lower surface.
(7) The waveguide substrate according to (6), wherein stray light absorption regions are provided on both upper and lower surfaces with the waveguide substrate interposed therebetween.
(8) The waveguide substrate according to (6) or (7), wherein the stray light absorption region is formed by adhesion of a light absorption member.
(9) The waveguide substrate according to any one of (6) to (8) above, wherein the incident light incident end face of the waveguide substrate is laser-cut.
(10) The waveguide substrate according to any one of (6) to (9), wherein a reaction vessel is formed by a light absorbing member.
(11) An evanescent wave excitation characterized in that it is a waveguide substrate used for evanescent wave excitation fluorescence observation having a reaction vessel with a test molecule fixed on the bottom, and the incident light incident end face is laser-cut. A waveguide substrate used for fluorescence observation.

According to the present invention, by providing the stray light absorption region at an effective position on the waveguide substrate, optically non-ideal stray light is absorbed before entering the reaction vessel, and the evanescent wave is excited by the slab type waveguide. When performing fluorescence observation, generation of background fluorescence, which is the biggest obstacle to observation, is suppressed, thereby enabling fluorescence observation with a high S / N ratio. Moreover, the stray light suppressing means of the present invention can be implemented easily and inexpensively, and in addition, it can be applied in combination with other methods for reducing the influence of stray light (liquid layer thickness control method), thereby further increasing the background fluorescence. Reduction is possible.

  The present invention relates to an evanescent wave excitation fluorescence observation method using a slide substrate itself as a slab type waveguide.

  As shown in FIG. 1, in the evanescent wave excitation light observation, the probe solution 14 is brought into contact with the waveguide substrate 5 on which the test molecule 19 is fixed, and then the binding reaction is sufficiently advanced, and then the incident light is guided. An evanescent wave 15 is generated on the waveguide by being incident from the end face of the waveguide substrate 5 and being repeatedly totally reflected in the substrate. Thereby, the fluorescence-labeled probe molecule 13 bound to the test molecule 19 emits fluorescence when excited by an evanescent wave, and the test molecule bound to the probe molecule on the substrate can be identified by detecting the fluorescence with the detector 16. .

  At this time, the intensity of the evanescent field that exudes from the interface has the property of exponentially decaying from the substrate interface in the vertical direction, and the region involved in the excitation of fluorescent molecules is about several hundreds of nanometers in the vertical direction from the interface. Therefore, even if the remaining probe solution layer is thick, in principle, the probe molecules 13 bound to the test molecules 19 on the waveguide substrate 5 are selectively detected. However, in an actual optical system, light (a broken line in FIG. 2) satisfying the total reflection condition among the light incident on the slide substrate serving as a waveguide travels inside the substrate, whereas the total reflection condition in the process of incidence or reflection. The background light is generated by directly exciting the fluorescently labeled probe molecules in the upper layer of the probe solution in which a part of the light (hereinafter referred to as stray light) that does not satisfy the conditions (solid line in FIG. 2) remains, and the S / N ratio decreases. As a result, a good image cannot be obtained with high sensitivity.

  On the other hand, the stray light suppressing means of the present invention is such that, in the waveguide substrate for evanescent wave excitation fluorescence observation, the width of the incident direction is at least on the waveguide substrate between the incident light incident end and the region where the test molecule is fixed. A stray light absorption region of 3 mm is provided.

  This stray light absorption region is provided only on the upper or lower surface of the substrate, or on both upper and lower surfaces, thereby supplementing stray light that escapes from the total reflection condition and escapes above the test molecule fixing region in the reaction tank. Yes (Figure 3). This suppresses the conventional process in which stray light generated in the optical system escapes upward in the test molecule fixing region in the reaction tank on the substrate, and the phenomenon that the fluorescent molecules in the upper layer of the probe solution are directly excited by stray light. Suppress. Further, by performing laser cutting on the incident end face of the incident light on the waveguide substrate, generation of scattered stray light on the end face is suppressed. For this reason, by using the substrate equipped with the stray light suppression method of the present invention, it becomes possible to significantly reduce the background light during the evanescent wave excitation fluorescence observation, and a good image with an improved S / N ratio can be obtained.

  As a means for forming the stray light absorption region of the present invention, for example, a means for applying a paint containing a black pigment such as carbon black, or a light absorbing member made of a molded product such as rubber or resin containing a black pigment, Means for bonding to the waveguide substrate and the like can be mentioned.

The form of the waveguide substrate provided with the light absorbing member of the present invention is shown in FIG. The light absorbing member of this form is designed to form the stray light absorbing region 6 by the upper and lower light absorbing members 3 in each reaction tank that is divided and formed by the upper waterproof partition material 1 on the waveguide substrate. Each light absorption member is provided in correspondence with the position of each reaction tank. The upper waterproof partition member 1 and the upper light absorbing member 3 can be bonded with an adhesive or the like. However, as shown in FIG. The upper light absorbing member may be integrally formed, and the reaction tank may be formed using the light absorbing member as a constituent member of the reaction tank side wall. Since this method can save the manufacturing cost of the upper waterproof partition member, it is a more advantageous method in terms of cost. The light absorbing member of the present invention can form the stray light absorbing region 6 having a width of at least 3 mm or more in the incident light direction on the waveguide substrate between the incident light incident end and the region where the test molecule is fixed. Any shape can be used.
Regarding the width of the stray light absorption region, as is clear from the experimental examples shown in the examples described later, if the width exceeds 5.4 mm, the background light reduction effect is not improved so much. The thickness of the waveguide substrate used in this experimental example is usually 1 mm. However, if the stray light absorption region having a width of at least 3 mm or 3 to 6.5 mm is provided, even if the thickness is larger than this, background light is provided. Can be effectively reduced.
As for the material of the light absorbing member disposed on the substrate, silicon rubber 20 ° (black) is used in this embodiment, but other materials (resin, sealing agent, etc.) that are in close contact with the slide glass may be used. Furthermore, the color is preferably black, but may be changed. The adhesive shape may also be adhered by the adhesiveness of the material itself, and an adhesive having as low autofluorescence as possible in the incident light wavelength region can be used. The thickness of the light-absorbing member layer is not particularly limited, but is usually preferably 1 mm or more, and may be made thicker for the convenience of operation. As a preferable example of such a waveguide substrate, the center distance in the vertical direction of the reaction tank disposed on the waveguide substrate is 8.4 to 9.6 mm, and this is disposed between the stray light absorption regions on the left and right sides of the substrate. (FIG. 8).

  Further, the arrangement method of the reaction tank is not limited to one horizontal row, and may be two or more horizontal rows. As an example, a system in which the reaction tanks are divided into two horizontal rows in the reaction tank area excluding the stray light absorption areas on the left and right sides of the substrate can be exemplified (FIG. 9). 8 and 9 are provided with stray light absorption regions of 5.4 mm on both the left and right sides in the longitudinal direction of the substrate.

  In addition, the waveguide substrate combined with the effect of reducing background light in the stray light suppression method of the present invention may be light-transmitting, but preferably has as little autofluorescence as possible and the end face shape is optically scattered light. It should be processed so that it has the characteristic that it is difficult to generate. Examples of such a plate-like material include borosilicate glass, white plate glass, quartz glass, synthetic quartz, and other optical use glasses (BK7, SF03, etc.). Further, as a more preferable form, in order to reduce generation of stray light due to scattering of incident light on the substrate end surface, use of a substrate subjected to laser cutting processing on the substrate end surface can be exemplified (FIG. 10). Furthermore, this substrate has the correspondence that can be observed with a scanner such as a confocal fluorescence observation scanner other than the evanescent wave excitation fluorescence scanner. After sufficiently reacting the probe sample solution with the immobilized molecules on the substrate, It is also possible to remove the light absorbing member and observe with a confocal fluorescence observation scanner or the like.

Moreover, you may improve intensity | strength and the operation convenience at the time of sample injection | pouring by providing an upper repair part decoration member and a lower modification member in the form superimposed on a light absorption member (FIG. 12). In addition, an upper protective seal can be provided to protect from physical contact from the top surface of the substrate and to prevent evaporation of the probe sample solution, and a lower protective seal can be used to prevent physical contact from the bottom surface of the substrate. Protection can be achieved.
FIG. 12 shows a case where the upper modifying member 8 and the upper protective seal 11 are disposed on the upper light absorbing member 3 and the lower modifying member 9 and the lower protective seal 12 are disposed below the lower light absorbing member 4. There is no particular inconvenience even if all or part of these modifying members and protective seals are not provided. However, when the upper modifying member 8 is provided on the upper light absorbing member 3 on the waveguide substrate 5 as shown in FIG. 13, the operability at the time of injecting the probe solution is greatly improved. Further, at this time, by providing the upper protective seal 11 on the upper light absorbing member 3, the convenience of handling is further improved by preventing evaporation of the probe sample solution. The lower protective seal 12 plays a role of preventing contamination of the back surface of the waveguide in the reaction tank due to physical contact from the lower surface by being attached to the lower modification member 9 or the lower light absorbing member 4. Further, the upper protective seal 11 and the lower protective seal 12 are made of a light-shielding material or a wavelength-selective light-transmitting material, thereby fading (photo bleaching) due to long-term light exposure of fluorescent dye molecules in the fluorescent probe solution. It is possible to provide a function to prevent.

Examples of the present invention are shown below, but the present invention is not limited to these Examples.

(1) Preparation of fluorescently labeled protein probe The fluorescently labeled protein probe has an absorption maximum wavelength around 550 nm of asialofetuine (hereinafter ASF) (SIGMA) or bovine serum albumin (BSA) (SIGMA). The fluorescent dye was prepared by fluorescent labeling using Cy3 Mono-reactive Dye (Amersham Pharmacia, hereinafter referred to as Cy3). As a procedure, after preparing ASF and BSA in phosphate buffered saline, pH 7.4 (hereinafter PBS) to a final concentration of 1 mg / mL, 1 mL is mixed with 1.0 mg of Cy3 powder for 1 hour. The reaction was carried out in the dark with timely stirring. Next, unreacted Cy3 molecules were removed by gel filtration chromatography using Sephadex G-25 (Amersham Pharmacia) as a carrier to purify as a Cy3-labeled protein probe preparation.

(2) Examining the installation position and optimum size width of the light absorbing member In order to design the shape of the black light absorbing member that absorbs stray light more effectively, the best balance between the spot area and the background light reduction effect is Knowledge of where is needed. For this study, we used a substrate with the same material and shape as the actual experimental system, and experimented under different conditions. Examples are shown below.
Cut the black silicon rubber 20 ° light-absorbing member (thickness 1 mm) punched to improve the adhesion surface smoothness, and make the distance between the absorbent material end and the slide end constant 6 mm. The reaction tank with the horizontal width of 2, 4, 6 mm changed to 3 stages, coated with 3-glycidoxypropyltrimethoxysilane (Shin-Etsu Silicone, hereinafter GTMS), 76.2 mm long x 25.4 mm wide It was fabricated on a white glass slide substrate having a thickness of mm (± 0.05 mm) and a thickness of 1.0 mm (± 0.05 mm). Next, 1 mL of purified water in which 100 ng / mL Cy3-BSA was dissolved was spotted on the back surface of the slide substrate and dried to prepare a spot sample for evaluation of fluorescence intensity by evanescent wave excitation. Next, fill the reaction tank with 100 μL of 1% BSA-containing PBS solution and leave it in a storage container kept at a humidity of 90% or more for 1 hour at 25 ° C. Probe sample molecules on the upper surface of the slide substrate in the reaction tank A blocking operation was carried out to suppress non-specific adsorption. 5 mg / mL of Cy3-BSA was added to each reaction tank on the substrate and left to stand for 1 hour.
As a result, it was observed that the background light decreased even with the same probe solution having the same volume as the width of the rubber in the incident direction of the incident light increased (FIG. 4). From this result, it was confirmed that the width of the rubber in the incident direction of the incident light is effective in reducing the background light. Also, in this experimental result, there was no significant change in the signal luminance value of the spot in each reaction tank. Therefore, in the reaction tank where the background light decreased, the entire excitation light did not decrease, but the upper layer of the probe solution. It can be seen that the background fluorescence from the source was selectively reduced.
In this experiment, under the present experiment conditions, the result was that the background light intensity decreased as the width of the light absorbing member in the incident direction of the incident light increased (FIG. 4). Furthermore, when this relationship was plotted, the correlation between the two was shown to have a curvilinear relationship (FIG. 5). In other words, it can be seen that the background reduction effect is not improved so effectively no matter how much the width is increased after the width of the light absorbing member in the incident light incident direction exceeds 5.4 mm. Based on these results, in this experimental system, the value of about 5.4 mm was derived for the width of the light absorbing member in the incident light incident direction as the best balance between the spot area and the background light reduction effect.

(3) Reduction of background light in evanescent wave excitation fluorescence observation
White glass waveguide, coated with 3-glycidoxypropyltrimethoxysilane (Shin-Etsu Silicone), 76.2 mm long × 25.4 mm wide (± 0.05 mm), 1.0 mm thick (± 0.05 mm) By repeating the dispensing operation of lectins on a substrate using a non-contact spotter, a protein array was prepared in which 43 spots of 43 proteins with a center-to-center distance of 3 spots were spotted. Here, a reaction tank forming rubber formed integrally with a light absorbing member made of black silicon rubber was adhered to form a reaction tank capable of filling eight liquids on the substrate. Next, fill the reaction tank with 100 μL of 1% BSA-containing PBS solution and leave it in a storage container kept at a humidity of 90% or more for 1 hour at 25 ° C. Probe sample molecules on the upper surface of the slide substrate in the reaction tank A blocking operation was carried out to suppress non-specific adsorption.
Add Cy3-ASF solution prepared at a concentration of 100 μg / mL, which is 1000 times higher than the normal use as a fluorescently labeled protein probe, to the microarray in the reaction vessel prepared through the above steps, and protect it from light. It was left to stand in an incubator set at 20 ° C. for 3 hours to react with the protein immobilized on the array. The slide substrate was observed under four different conditions to verify the effect of the stray light suppression method. When the light absorbing member was attached to the slide substrate, an experiment was performed to compare the difference in background light between the case where only the upper surface was attached and the case where the light absorbing member was attached to the upper and lower surfaces. Using the slide after probing the entire reaction vessel with 100 ng / mL Cy3-ASF probe, the probe solution in the reaction vessel was replaced with a Cy3-glycine solution, and then scanned. For scanning, an evanescent wave excitation type microarray scanner (GTMAS Scan IV) was used, and evanescent wave excitation fluorescence observation was performed. Scanning parameters were Gain “4000 times”, integration number “4 times”, and exposure time “34 msec”. Array-Pro Analyzer (version 4.0 for Windows, Media Cybernetics), a commercially available analysis software for microarrays, was used to digitize the brightness of the scanned image.
As a result, when the intensity of the background light of the upper layer of the probe solution was observed with the absorbent material attached only on the upper surface and the upper and lower surfaces, the method of attaching the light absorbing member to the upper and lower surfaces of the slide substrate was the upper surface. Compared with the method of attaching only to the substrate, the effect of greatly reducing the fluorescence derived from the upper layer of the probe solution was high, and the result was more effective for background light removal (FIG. 10).

(4) Observation of effect when combined use of stray light suppression method and liquid layer thickness control method For a system in which background light derived from the probe solution upper layer is suppressed using a light absorbing member, a borosilicate glass plate is used as a liquid layer thickness control member in the reaction vessel By setting the inside of the probe solution, the background light intensity of the upper layer of the probe solution was observed when the stray light suppression method of the present invention and the liquid layer thickness control method (Japanese Patent Application No. 2006-203257) which was the background light reduction method previously applied were used in combination. . Remove the probe solution of the substrate probed with 100 ng / mL Cy3-ASF from the reaction vessel, and deliberately add a high concentration of fluorescent-labeled glycoprotein probe solution (10 μg / mL Cy3-BSA). The resulting solution was injected into a 100 μL reaction vessel and observed with an evanescent wave excitation fluorescence scanner. As a result, a further background light attenuation effect was observed by controlling the liquid layer thickness using a liquid layer thickness controller for the system in which the background light derived from the probe solution upper layer was suppressed by the stray light suppression method. From this experimental result, it was found that it is possible to use it together with the liquid layer thickness control device by attaching the absorbent to the lower surface of the substrate, and both methods enhance the effect of attenuating background fluorescence derived from the upper layer of the probe solution. (FIG. 11).

It is a figure which shows the principle of the selective excitation of the solid-liquid interface vicinity in the conventional evanescent wave excitation fluorescence observation. It is the figure which showed the board | substrate used when performing the conventional evanescent wave excitation fluorescence observation, and its characteristic. It is the figure which showed the characteristic of the waveguide board | substrate provided with the stray light suppression means used for the evanescent wave excitation fluorescence observation invented this time. It is a figure which shows the result of having observed the relationship between the width | variety of the light absorption member of the incident direction of incident light, and the fluorescence intensity of an upper layer. It is a figure which shows the relationship between the width | variety of the light absorption member of the incident direction of incident light, and the fluorescence intensity of an upper layer. It is a figure which shows the arrangement | positioning form of the member of the board | substrate which comprised the light absorption member in the stray light absorption area | region on the waveguide board | substrate which provided the reaction tank of this invention. It is a figure which shows the arrangement | positioning form of the member of the board | substrate which comprised the light absorption member in the stray light absorption area | region on the waveguide board | substrate which provided the reaction tank of this invention. It is a figure which shows the dimension of the light absorption member designed in the case of implementation of this invention. It is a figure which shows the dimension of the light absorption member designed in the case of implementation of this invention. It is a figure which shows the result of the experiment which compared the background light attenuation effect of the stray light absorption method of this invention with the background light attenuation effect of the conventional board | substrate. It is a figure which shows the experimental result which observed the background light reduction effect by the combination of a stray light absorption method and a liquid layer thickness control method. It is a figure which shows the arrangement | positioning form of the attached modifier to the board | substrate which comprised the light absorption member in the stray light absorption area | region on the waveguide board | substrate which provided the reaction tank of this invention. It is a figure which shows the more preferable usage pattern which attached the attachment modifier to the board | substrate which comprised the light absorption member in the stray light absorption area | region on the waveguide board | substrate which provided the reaction tank of this invention.

Claims (9)

A probe solution containing a fluorescently labeled probe molecule is introduced into a reaction vessel having a waveguide substrate with a test molecule fixed to the bottom, and the fixed molecule and the probe molecule are bonded together, and then from the end face of the waveguide substrate When performing evanescent wave excitation fluorescence observation by introducing light, it is a method for suppressing background light in evanescent wave excitation fluorescence observation by suppressing the incidence of stray light to the reaction vessel,
On surfaces of the waveguide substrate while the incident light entering end reaches the test molecule fixing region, it provided the stray light absorption regions having at least 3mm width in the incident light direction,
The light-absorbing member forming the stray light absorption regions, and characterized by forming a side wall of the reactor, background light reduction method. The background light reducing method according to claim 1, wherein the stray light absorption regions are provided on both upper and lower surfaces with the waveguide substrate interposed therebetween. The background light reducing method according to claim 1, wherein the stray light absorbing region is formed by adhesion of a light absorbing member. The incident light incident end face of the waveguide substrate is characterized in that it is a laser cutting process, the background light reduction method of any one of claims 1 to 3. The background light reduction method according to claim 1, wherein the test molecule at the bottom of the reaction vessel is a microarray test molecule . A waveguide substrate used for observation of evanescent wave-excited fluorescence, having a reaction vessel with a test molecule fixed at the bottom,
On surfaces of the waveguide substrate between reaching the test molecule fixing area from the entrance end of the light incident from the end face of the waveguide substrate, the stray light absorbing region having at least 3mm or more widths are provided in the incident direction,
A waveguide substrate , wherein a side wall of the reaction vessel is formed by a light absorbing member that forms the stray light absorbing region . Characterized in that the stray light absorption regions are provided on both upper and lower surfaces across the waveguide substrate, a waveguide substrate according to claim 6. The waveguide substrate according to claim 6 or 7, wherein the stray light absorption band is formed by adhesion of a light absorbing member. Wherein the incident light incident end face of the waveguide substrate are laser cutting process, waveguide substrate of any one of claims 6-8.