CN115308170A - Fluorescence image detection system and fluorescence detection method - Google Patents
Fluorescence image detection system and fluorescence detection method Download PDFInfo
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- CN115308170A CN115308170A CN202210481623.XA CN202210481623A CN115308170A CN 115308170 A CN115308170 A CN 115308170A CN 202210481623 A CN202210481623 A CN 202210481623A CN 115308170 A CN115308170 A CN 115308170A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention provides a fluorescence image detection system for detecting a fluorescence image of an object to be detected. The fluorescence image detection system comprises an excitation light source, a surface scanning image acquisition device, a filtering module and a controller. The excitation light source generates excitation light to at least one irradiation area on the object to be detected, so that the object to be detected generates fluorescence. The surface scanning image acquisition device receives the fluorescence from the object to be detected so as to acquire a fluorescence image of the object to be detected. The light filtering module is coupled between the surface scanning image acquisition device and the object to be detected to filter the exciting light and enable the fluorescence to pass through the light filtering module. The controller is coupled to the area scan image acquisition device to receive and detect the fluorescence image. A fluorescence detection method is also provided.
Description
Technical Field
The present invention relates to a detection system and a detection method, and more particularly, to a fluorescence image detection system and a fluorescence detection method.
Background
In conventional visible light detection techniques, materials with high reflectivity or high transmittance are not easily well detected by visible light. For example, the material of high reflectivity such as metal exists in the object to be detected, so that the detection result is easily misjudged. Alternatively, the transparent colloid is not readily visibly present in the visible light detection.
Fluorescence is a phenomenon in which an object absorbs excitation light having a short wavelength and emits emission light having a long wavelength. Fluorescence detection utilizes organic matter in an object to be detected to generate fluorescence, which can provide higher detection quality, and thus becomes one of the options of Automatic Optical Inspection (AOI). For example, the transparent colloid can clearly highlight the appearance through the fluorescence generated after the colloid is irradiated with the excitation light.
However, since fluorescence is a rather weak light source, it is difficult to mass-produce under the high throughput requirements of industrial detection. In addition, if the irradiation intensity or time of the excitation light is increased in order to increase the intensity of the fluorescence, the effect of Photobleaching (Photobleaching) is caused, and the analyte is damaged.
Disclosure of Invention
The invention provides a fluorescence image detection system and a fluorescence detection method, which can improve the fluorescence detection effect under the condition of avoiding photobleaching.
An embodiment of the invention provides a fluorescence image detection system for detecting a fluorescence image of an object to be detected. The fluorescence image detection system comprises an excitation light source, a surface scanning image acquisition device, a filtering module and a controller. The excitation light source generates excitation light to at least one irradiation area on the object to be detected, so that the object to be detected generates fluorescence. The surface scanning image acquisition device receives the fluorescence from the object to be detected so as to acquire a fluorescence image of the object to be detected. The light filtering module is coupled between the surface scanning image acquisition device and the object to be detected to filter the exciting light and enable the fluorescence to pass through the light filtering module. The controller is coupled to the area scan image acquisition device to receive and detect the fluorescence image.
One embodiment of the present invention provides a fluorescence detection method, which includes the following steps. At least one irradiation area is defined on the object to be measured based on the object to be measured. And correspondingly setting an energy accumulation threshold of at least one irradiation region according to at least one irradiation region. At least one irradiation area on the object to be measured is irradiated by the excitation light source, so that the object to be measured generates fluorescence. The analyte is detected by image detection through fluorescence. When the energy accumulated in at least one irradiation area reaches an energy accumulation threshold value, the irradiation of the irradiation area by the excitation light source is stopped.
Based on the above, in an embodiment of the invention, since the fluorescence image detecting system and the fluorescence detecting method control the time of the irradiation region of the object to be detected irradiated with the excitation light according to the energy accumulation threshold, the fluorescence image detecting system and the fluorescence detecting method can improve the fluorescence detecting effect while avoiding the light-induced color fading. Moreover, because the large-area array surface scanning image acquisition device is used, compared with the line array image sensor, the fluorescent image detection system provided by the embodiment of the invention can meet the requirement of high-speed detection.
Drawings
FIG. 1 is a schematic optical path diagram of a fluorescence image detection system according to an embodiment of the present invention;
FIG. 2 is a perspective view of a fluorescence image detection system according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of multiple illumination regions for an object under test;
FIG. 4 is a flow chart of a fluorescence detection method according to an embodiment of the invention.
Detailed Description
Referring to fig. 1-3, a fluorescence image detection system 100 according to an embodiment of the invention is used for detecting a fluorescence image of an object S. The fluorescence image detection system 100 includes an excitation light source 110, a surface scanning image acquisition device 120, a filter module 140A, and a controller 130. In the present embodiment, the object S includes, but is not limited to, a printed circuit board, a wafer or other objects containing organic matters, and the organic matters include transparent glue on the printed circuit board or contaminants on the wafer.
In the present embodiment, the excitation light source 110 is used to emit excitation light EL. The excitation light source 110 may be a light-emitting Diode (LED) light source, a Laser Diode (Laser Diode) light source, or other suitable light sources. The excitation light EL may be ultraviolet light, blue light, white light, or other color light. In this embodiment, the excitation light source 110 generates excitation light EL to at least one irradiation region IR of the object S to generate fluorescence F from the object S. For example, the object S may convert uv light into blue light or may convert blue light into green light, but the invention is not limited thereto.
In the present embodiment, the area scan image obtaining device 120 receives the fluorescence F from the object S to obtain a fluorescence image of the object S. The area scan image capturing device 120 may be a photosensor such as a Complementary Metal-Oxide Semiconductor (CMOS), a Charge Coupled Device (CCD), or a photodiode (photodiode), but the invention is not limited thereto.
In one embodiment, the controller 130 includes, for example, a Central Processing Unit (CPU), a microprocessor (microprocessor), a Digital Signal Processor (DSP), a programmable controller, a Programmable Logic Device (PLD), or other similar devices or combinations thereof, which are not limited in the present invention. Furthermore, in one embodiment, the functions of the controller 130 may be implemented as a plurality of program codes. The program codes are stored in a storage unit and executed by the controller 130. Alternatively, in one embodiment, the functions of the controller 130 may be implemented as one or more circuits. The present invention is not limited to the implementation of the functions of the controller 130 in software or hardware.
In this embodiment, the controller 130 is coupled to the excitation light source 110 or to the area scan image acquisition device 120 to receive and detect the fluorescence image. The controller 130 controls the excitation light source 110 to continuously emit the excitation light EL within the energy accumulation threshold, and stops irradiating the irradiation region IR with the excitation light source 110 when the energy accumulated in the irradiation region IR reaches the energy accumulation threshold. In the present embodiment, the irradiation of the irradiation region IR with the excitation light EL is stopped by, for example, turning off the excitation light source 110. Alternatively, an aperture is provided in the transmission path of the excitation light EL, and the controller 130 controls the aperture to cause the irradiation region IR to be irradiated with the excitation light EL or to stop being irradiated.
In one embodiment, the sample S is a photoresist, including but not limited to, an organic material such as a phenolic resin. The irradiation region IR is, for example, 10mm on the object S to be measured 2 A region of size. Irradiating the irradiation region IR with near ultraviolet light as excitation light EL to accumulate energy to 50mj/mm 2 Photobleaching occurs afterwards. If the excitation light EL is at 10mj/mm per second 2 The intensity is irradiated to the irradiated area IR, and this energy accumulation may correspond to 5 seconds. That is, the organic matter on the irradiation region IR of the object S may be photo-discolored after the cumulative irradiation for about 5 seconds. In this embodiment, including but not limited to, 80% of the energy at which photobleaching occurs can be set as the threshold for energy accumulation.
In the present embodiment, the pixels of the area scan image acquisition device 120 are, for example, 14,000 × 10,000; therefore, compared with the conventional line scan, the area scan image capturing apparatus 120 has 10000 times of exposure time. In one embodiment, the total pixels of the area scan image capture device 120 may be an upper limit of the process. Moreover, the signal-to-noise ratio can be improved by increasing the exposure time of the area scan image capturing device 120, for example, the exposure time of the area scan image capturing device 120 can be increased to be equal to or greater than the time corresponding to the energy accumulation threshold. Therefore, in the case that the fluorescence is usually a weak light source, by using the area scanning image acquisition device with the large area array and by increasing the exposure time, the fluorescence image detection system 100 according to the embodiment of the present invention can meet the requirement of high-speed detection compared to using the line array image sensor.
In the present embodiment, the excitation light source 110 includes a plurality of light guide fiber light sources with different illumination directions. As shown in fig. 2, the excitation light EL is irradiated onto the object S to be measured in four diagonal directions by the light guide fiber light source. Thus, a uniform light source can be provided, and the energy required for supplying fluorescence can be increased. In another embodiment, the excitation light source may comprise a ring-type light source.
In the present embodiment, the fluorescence image detection system 100 further includes a filter module 140A. The filtering module 140A is coupled between the surface-scan image obtaining device 120 and the object S to be measured, and is used for filtering the excitation light EL and making the fluorescence F pass through the filtering module 140A. In one embodiment, the fluorescence image detection system 100 further includes another filter module 140B. The filtering module 140B is coupled between the excitation light source 110 and the object S on a transmission path of the excitation light EL, so that the excitation light EL passes through and the rest of the color light is filtered.
In one embodiment, the fluorescence image detection system 100 further includes a beam splitter 200. The spectroscope 200 is used to reflect the excitation light EL and transmit the fluorescence F. The beam splitter 200 is coupled between the filter module 140B and the object S on the transmission path of the excitation light EL, and is coupled between the filter module 140A and the object S on the transmission path of the fluorescence F.
In one embodiment, the fluorescent image detection system 100 further includes an objective lens 300. The beam splitter 200 is disposed between the objective lens 300 and the filtering module 140A.
In the present embodiment, the fluorescent image detecting system 100 further includes a zoom lens 400. The controller 130 is coupled to the zoom lens 400, so that the fluorescence image detection system 100 can adjust the magnification of the lens according to the type of the object S.
In this embodiment, the fluorescence image detection system 100 further includes a mobile stage 500. The controller 130 is coupled to the movable stage 500, so that the fluorescence image detection system 100 can adjust the relative position between the fluorescence image detection system 100 and the object S, and further adjust the position of the irradiation region IR.
In the present embodiment, the fluorescent image detection system 100 further includes a front focusing module 600. The leading focusing mode is to detect the Z depth of the N +1 position in the N position before measuring the focusing of the N +1 position, and adjust the focusing of the Z depth when the camera moves from the N position to the N +1 position to reduce the focusing time for finding the next position. The controller 130 is coupled to the front focusing module 600, so that the fluorescent image detecting system 100 can respond to the correct focusing plane in real time through focusing feedback, thereby improving the detection quality and reducing the detection time.
Referring to fig. 4, a fluorescence detection method according to an embodiment of the invention includes the following steps. Step S100 is to define at least one irradiation region IR on the object S based on the object S. Step S110, correspondingly setting an energy accumulation threshold of at least one irradiation region IR according to at least one irradiation region IR. Step S120, the excitation light source 110 irradiates at least one irradiation region IR on the object S to be measured, so that the object S to be measured generates fluorescence F. In step S130, the specimen S is detected by image detection through the fluorescence F. When the energy accumulated in at least one irradiation region IR reaches the energy accumulation threshold, the irradiation of the irradiation region IR with the excitation light source 110 is stopped.
In this embodiment, the fluorescence detection method further includes the following steps. The unit of illumination energy provided by the excitation light source 110 is corrected. For example, the unit irradiation energy (e.g., joules/second or mjoules/second) of the excitation light source 110 is first determined by an energy detector.
In this embodiment, the step of irradiating the at least one irradiation region IR on the object S with the excitation light source 110 includes: recording the energy accumulation status of at least one irradiation area IR.
In this embodiment, the step of setting the energy accumulation threshold of at least one irradiation region IR includes: and setting an energy accumulation threshold value according to the material type of at least one irradiation region IR.
In summary, in an embodiment of the invention, since the fluorescence image detection system and the fluorescence detection method control the time for the excitation light to irradiate the object to be detected according to the established irradiation region and the energy accumulation threshold corresponding to the irradiation region, the fluorescence image detection system and the fluorescence detection method can improve the fluorescence detection effect while avoiding the light-induced color fading. Moreover, because the large-area array surface scanning image acquisition device is used, compared with the line array image sensor, the fluorescent image detection system provided by the embodiment of the invention can meet the requirement of high-speed detection.
Claims (13)
1. A fluorescence image detection system for detecting a fluorescence image of an object to be detected, comprising:
the excitation light source is used for generating excitation light to at least one irradiation area on the object to be detected so as to enable the object to be detected to generate fluorescence;
a surface scanning image acquisition device for receiving the fluorescence from the object to be detected to obtain the fluorescence image of the object to be detected;
the light filtering module is coupled between the surface scanning image acquisition device and the object to be detected so as to filter the exciting light and enable the fluorescence to pass through the light filtering module; and
a controller coupled to the area scan image acquisition device to receive and detect the fluorescence image.
2. The fluorescent image detection system according to claim 1, further comprising:
another filtering module, coupled between the excitation light source and the object to be measured on the transmission path of the excitation light, so as to allow the excitation light to pass through and the rest color light to be filtered; and
and the spectroscope is coupled between the other filter module and the object to be detected on the transmission path of the excitation light so as to reflect the excitation light and transmit the fluorescence.
3. The fluorescent image detection system of claim 1, further comprising a lead focus module coupled to the controller.
4. The fluorescence image detection system of claim 1, wherein the object to be detected comprises a printed circuit board, a wafer, or other object containing organic matter.
5. The fluorescence image detection system according to claim 1, wherein the irradiation of the at least one irradiation region with the excitation light source is stopped when the accumulated energy of the at least one irradiation region reaches an energy accumulation threshold.
6. The fluorescence image detection system according to claim 1, wherein the excitation light source includes a plurality of light guide fiber light sources or ring light sources with different illumination directions.
7. The fluorescent image detecting system according to claim 1, wherein the total pixels of the area scan image obtaining means are 14,000 x 10,000 or more.
8. The fluorescence image detection system according to claim 5, wherein the exposure time of the area scan image acquisition device is equal to or longer than the time corresponding to the energy accumulation threshold.
9. The fluorescence image detection system of claim 5, wherein the energy accumulation threshold is 80% of a time that the test object undergoes photobleaching within the at least one illuminated area.
10. A method of fluorescence detection, comprising:
defining at least one irradiation area on an object to be detected based on the object to be detected;
correspondingly setting an energy accumulation threshold of the at least one irradiation region according to the at least one irradiation region;
irradiating the at least one irradiation area on the object to be detected by using an excitation light source to enable the object to be detected to generate fluorescence; and
detecting the object to be detected through the fluorescence and images;
wherein the irradiation of the at least one irradiation region with the excitation light source is stopped when the accumulated energy of the at least one irradiation region reaches the energy accumulation threshold.
11. The fluorescence detection method according to claim 10, further comprising:
and correcting the unit irradiation energy provided by the excitation light source.
12. The fluorescence detection method according to claim 10, wherein the step of irradiating the at least one irradiation region on the analyte with the excitation light source includes:
recording an energy accumulation state of the at least one irradiation region.
13. The fluorescence detection method of claim 10, wherein the step of setting the energy accumulation threshold for the at least one illumination region comprises:
and setting the energy accumulation threshold value according to the material type of the at least one irradiation area.
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| TW110116556A TWI830022B (en) | 2021-05-07 | 2021-05-07 | Fluorescence image detection system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117147586A (en) * | 2023-10-26 | 2023-12-01 | 江苏纳沛斯半导体有限公司 | COF resin region foreign matter detection method |
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| JP4673000B2 (en) * | 2004-05-21 | 2011-04-20 | 株式会社キーエンス | Fluorescence microscope, display method using fluorescence microscope apparatus, fluorescence microscope image display program, computer-readable recording medium, and stored device |
| US10317347B2 (en) * | 2013-11-01 | 2019-06-11 | Kla-Tencor Corp. | Determining information for defects on wafers |
| TWM479415U (en) * | 2014-01-15 | 2014-06-01 | meng-da Guo | Fluorescence automatic optical inspection machine |
| DE102016122529A1 (en) * | 2016-11-22 | 2018-05-24 | Carl Zeiss Microscopy Gmbh | Microscope for imaging an object |
| CN108982454B (en) * | 2018-07-30 | 2021-03-02 | 华中科技大学苏州脑空间信息研究院 | Axial multilayer parallel scanning microscopic imaging method and system |
| CN109238965A (en) * | 2018-09-25 | 2019-01-18 | 军事科学院系统工程研究院卫勤保障技术研究所 | A kind of dry chemical blood cell composition analysis optical system |
| CN111272715B (en) * | 2018-12-04 | 2023-03-14 | 长春长光华大智造测序设备有限公司 | Fluorescence imaging system of gene sequencer |
| CN112525870B (en) * | 2019-09-17 | 2023-07-04 | 北京达微生物科技有限公司 | Large-area fluorescence imaging detection device |
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| CN117147586A (en) * | 2023-10-26 | 2023-12-01 | 江苏纳沛斯半导体有限公司 | COF resin region foreign matter detection method |
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