Apparatus and method for reading fluorescence

A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising:

• a substrate 2 to which a probe substance 1 is fixed;
• a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
• a light source 7;
• an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field;
• and
• a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
• wherein the optical system 8 comprises an incidence angle adjusting means 10 for adjusting an incidence angle when the light from the light source 7 is incident on the substrate 2;
• and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10,
• wherein the controller 11 comprises a means for receiving information on the incidence angle adjusted by the incidence angle adjusting means 10 and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles detected by the fluorescence detecting unit 9 and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles;
• and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths;
• and
• wherein the substrate 2 comprises a spot 21 to which the probe substance 1 is fixed;
• wherein the fluorescence detecting unit 9 obtains the fluorescence image generated by the fluorescent substance 3;
• and
• wherein the controller 11 comprises a means for scanning the fluorescence image to compute a boundary 21 at which the fluorescence intensity is changed;
• a means for grasping a region 23 inside the spot and a region 24 outside the spot in the fluorescence image from the shape of the boundary;
• and a means for obtaining the fluorescence intensity inside the spot.

A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising:

• a substrate 2 to which a probe substance 1 is fixed;
• a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
• a light source 7;
• an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field;
• and
• a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
• wherein the optical system 8 comprises an incidence angle adjusting means 10 for adjusting an incidence angle when the light from the light source 7 is incident on the substrate 2;
• and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10;
• and
• wherein the controller 11 comprises a means for receiving information on the incidence angle adjusted by the incidence angle adjusting means 10 and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles detected by the fluorescence detecting unit 9 and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles;
• and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths;
• and
• wherein the concentration of the probe substance 1 is increased toward the downstream region of the sample chamber in the substrate 2 to which the probe substance is fixed.

A fluorescence reading method using a fluorescence reading apparatus that detects a specific interaction between a probe substance and a target substance, comprising:

• a substrate 2 to which a probe substance 1 is fixed;
• a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
• a light source 7;
• an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field;
• and
• a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
• characterized in that a step of changing the penetration depth of the evanescent field and a step of obtaining the fluorescence intensity after the penetration depth of the evanescent field is changed are repeatedly performed to obtain the fluorescence intensities in a plurality of penetration depths of the evanescent fields,
• wherein a graph is obtained where one axis represents the fluorescence intensity while the other axis represents the penetration depth of the evanescent field, using the relationships between the penetration depths of the evanescent fields and the fluorescence intensities with respect to the plurality of penetration depths.

A fluorescence reading apparatus 12 that detects a specific interaction between a probe substance and a target substance, comprising:

• a substrate 2 to which a probe substance 1 is fixed;
• a sample chamber 6 that accommodates the probe substance 1 and also accommodates a sample 5 that contains a fluorescent substance 3 and a target substance 4 such that the sample is brought into contact with the probe substance 1;
• a light source 7;
• an optical system 8 that guides the light from the light source 7 to the substrate 2 to generate an evanescent field;
• and
• a fluorescence detecting unit 9 that detects a fluorescence intensity or a fluorescence image generated by the fluorescent substance 3 excited by the evanescent field,
• characterized in that the concentration of the probe substance 1 is increased toward the downstream region of the sample chamber in the substrate 2 to which the probe substance is fixed.