CN110974173A - Fluorescence imaging system for experimental animals - Google Patents
Fluorescence imaging system for experimental animals Download PDFInfo
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Abstract
The invention discloses a fluorescence imaging system for experimental animals, which comprises a light source light path and a fluorescence imaging light path; the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the light source circuit also comprises a coupling optical fiber bundle and a plurality of light emitting units for respectively generating fluorescence excitation light or optogenetic stimulation light with different wavelengths; the coupling optical fiber bundle comprises a plurality of optical fibers, wherein the light outlet ends of the optical fibers are closely arranged, and the light inlet ends of the optical fibers are divided into a plurality of sub-beams; the light-incoming ends of the sub-beams are respectively coupled with the light-emitting units in a matching way; the optical fibers of the plurality of sub-beams are arranged in the coupling optical fiber bundle in a mixed manner, and the distance from the light outlet end to the rear end face of the objective lens is within 2.5 times of the focal length; the fluorescence imaging optical path also comprises an imaging unit and an image acquisition unit; the invention has small volume, light weight, good imaging effect and easy expansion, and can realize multicolor fluorescence imaging and in-situ optical genetic fluorescence imaging.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a wearable multi-light-source fluorescence imaging system for experimental animals.
Background
The detection of the neuron activity signals is the basis of neuroscience research, and conventional means such as electrophysiological recording have the problems of high technical difficulty, low flux, no specificity, lack of spatial resolution and the like. The development of optical nerve probes such as calcium sensitive fluorescent probes, neurotransmitter sensitive probes and voltage sensitive probes makes it possible to apply the optical imaging technology to the detection and recording of nerve activities, well makes up for the defects of the traditional technology, and is an important supplement to the modern neuroscience research and detection technology.
Wide field fluorescence microscopy is an important tool for optical imaging of neuronal activity. The wide-field fluorescence microscope can be used for monochromatic and multicolor (such as double-layer and three-color) neuron activity fluorescence imaging by matching with corresponding fluorescence probes, and can also be used for performing in-situ optogenetic stimulation simultaneously by adding optogenetic stimulation light sources. Miniaturized wide-field fluorescence microscopes, which are implemented by using micro-optics, micro-imaging elements and micro-mirror structures, are ideal solutions for optical imaging of free-moving animals in somatic nerves, and have now begun to be applied in neuroscience research at home and abroad. However, the light source adopted by the existing micro fluorescence microscope is that a light emitting diode is assembled on a microscope body to serve as a self-contained light source, and a collimating lens and a narrow-band filter are required to be used in a matched manner (such as the invention patent of China with the application number of 201910621446.9), so that the structure is complex, the cost is high, the assembly difficulty is high, and the light source replacement relates to the microscope disassembly, and the light wavelength is difficult to replace according to the actual research requirement. Particularly, if the wavelength of light is increased to perform multicolor imaging or increase the optogenetic function, each time one wavelength of light is increased, a set of light source consisting of a light emitting diode, a collimating lens and a narrow-band filter needs to be added, and a dichroic mirror for guiding the light source needs to be additionally added, so that the light path is complicated, and the increase to more than two wavelengths of light is difficult. Although the light wavelength can be increased to two light wavelengths, the increased components and space of the lens body can cause the lens body to have large volume, heavy weight, complex assembly and high cost, and because the coupling efficiency of the light-emitting diode is low, the light-emitting diode needs to use larger power when carrying out higher-energy optogenetic research, generates heat seriously and is not suitable for continuous experiments worn on the head of an animal for a long time.
Therefore, in order to solve the problems in the prior art and meet the requirements of the present neuroscience on the research of multicolor fluorescence imaging and in-situ optogenetic stimulation-fluorescence imaging on freely moving animals, a multifunctional micro fluorescence microscope which can be expanded into a plurality of light source wavelengths, has small volume, light weight, simple structure and easy assembly needs to be developed.
Disclosure of Invention
Aiming at the defects or the improvement requirements of the prior art, the invention provides a wearable multi-light-source fluorescence imaging system for experimental animals, which aims to externally arrange a light source of the imaging system and simplify an illumination light path through multi-fiber coupling light beams which are arranged in a mixed mode, so that the weight of the wearable system is reduced, the luminous capacity of the light source is expanded, and the technical problem that the activity of the experimental animals is influenced due to single function, large volume and heavy weight of the conventional wearable fluorescence imaging system is solved.
To achieve the above object, according to one aspect of the present invention, there is provided a wearable multi-light-source fluorescence imaging system for experimental animals, comprising a light source optical path and a fluorescence imaging optical path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue;
the fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the light source circuit also comprises a coupling optical fiber bundle and a plurality of light emitting units for respectively generating fluorescence excitation light or optogenetic stimulation light with different wavelengths; the coupling optical fiber bundle comprises a plurality of optical fibers, wherein the light outlet ends of the optical fibers are closely arranged, and the light inlet ends of the optical fibers are divided into a plurality of sub-beams; the light-incoming ends of the sub-beams are respectively coupled with the light-emitting units in a matching way; the optical fibers of the plurality of sub-beams are arranged in a hybrid manner in the coupling optical fiber bundle, so that the light spots from the plurality of sub-beams coincide at the light outlet end, and in-situ multicolor imaging or in-situ light genetic stimulation-fluorescence imaging is realized; the distance between the light-emitting surface of the optical fiber bundle and the rear end surface of the objective lens is within 2.5 times of the focal length; the fluorescence imaging light path further comprises an imaging unit and an image acquisition unit;
the light emitting unit of the light source light path generates fluorescence excitation light and/or stimulation light with different wavelengths, the fluorescence excitation light and/or the stimulation light are respectively coupled to corresponding sub-beams, meanwhile, the fluorescence excitation light and/or the stimulation light are transmitted to the dichroic mirror through the optical fiber beams, and the reflected fluorescence excitation light and/or the stimulation light are projected to a target area through the objective lens; the fluorescence emitted by the target area is collected by the same objective lens and is transmitted through the dichroic mirror, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
Preferably, the wearable multi-light source fluorescence imaging system for experimental animals has the coupling fiber bundle diameter of 0.3-3.0 mm and the sub-bundle fiber density of 11-110 fibers per square mm.
Preferably, the wearable multi-light-source fluorescence imaging system for experimental animals has the advantages that the light intensity fluctuation in the observation range of the visual field of the light spot formed by each sub-beam of the coupled optical fiber bundle is not more than 10%.
Preferably, the wearable multi-light-source fluorescence imaging system for experimental animals has the light outlet end of the coupled optical fiber bundle wrapped by a section of rigid sleeve and fixed relative to the fluorescence imaging optical path.
Preferably, in the wearable multi-light-source fluorescence imaging system for experimental animals, a light uniformizing device is arranged at a light emitting end of the coupling optical fiber bundle, and the light uniformizing device comprises a light uniformizing sheet, light dispersion glue and a large-core-diameter coupling optical fiber.
Preferably, the optical fiber of the wearable multi-light-source fluorescence imaging system for experimental animals is a plastic optical fiber, so that high flexibility is realized, meanwhile, the wearable multi-light-source fluorescence imaging system is not easy to break and damage, and is suitable for research of free-moving animals.
Preferably, the wearable multi-light-source fluorescence imaging system for experimental animals comprises a light source and a driving module thereof, wherein the light source is a high-power LED-narrow-band filter combination or an external laser; preferably, the driving module is configured to modulate the light source according to an external TTL signal.
Preferably, in the wearable multi-light-source fluorescence imaging system for experimental animals, the dichroic mirror is a multi-cut-off wavelength dichroic mirror, and is configured to reflect fluorescence excitation light and/or stimulation light of different wavelengths generated by the light emitting unit of the light source light path at the same time.
Preferably, the wearable multi-light-source fluorescence imaging system for experimental animals, the imaging unit of the fluorescence imaging optical path thereof, comprises an imaging lens, preferably a fluorescence filter, and the filter is preferably between the dichroic mirror and the imaging lens; the image acquisition unit of the fluorescence imaging light path comprises an array image detector and a peripheral circuit thereof, and the array image detector is preferably a multicolor array image detector.
Preferably, the wearable multi-light-source fluorescence imaging system for experimental animals comprises a mirror body for setting the light source light path and the fluorescence imaging light path, wherein the mirror body is made of a rigid light-tight high polymer material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
the wearable multi-light-source fluorescence imaging system for experimental animals provided by the invention adopts the coupling optical fiber bundle to guide the fluorescence excitation light or the stimulation light generated by the light-emitting unit to enter the fluorescence imaging system and simultaneously plays a plurality of roles: firstly, the weight of the mirror body is reduced due to the external light source, and the influence on the movement of an experimental animal is small; secondly, except a dichroic mirror, other optical devices including a collimating lens and an optical filter are not arranged between the light outlet end of the optical fiber bundle and the objective lens, so that the weight of the lens body is further reduced, the volume of the lens body is reduced, and the quality of illumination light is improved; thirdly, emergent light of the coupled light beams formed by the tight arrangement of multiple optical fibers is overlapped to form platform-like light spots, so that Gaussian light spots are avoided, the light spot quality of fluorescence excitation light and optical genetic spine laser is improved, and a better imaging effect can be obtained; and fourthly, the light inlet ends of the multiple optical fibers can be freely combined or expanded into a plurality of independent light guide sub-beams, and different light sources are respectively coupled according to different conditions, so that multicolor fluorescence imaging, in-situ optogenetic imaging and even multicolor fluorescence in-situ optogenetic imaging are realized. In summary, the invention has small volume, light weight, good imaging effect and easy expansion, and can realize multicolor fluorescence imaging and in-situ optical genetic fluorescence imaging.
Drawings
FIG. 1 is a schematic structural diagram of a wearable multi-light-source fluorescence imaging system provided by the present invention;
FIG. 2 is a schematic external view of a wearable multi-light-source fluorescence imaging system provided by the present invention;
FIG. 3 is a schematic view of light source coupling of a wearable multi-light-source fluorescence imaging system provided in embodiment 1 of the present invention;
FIG. 4 is a schematic view of light source coupling of a wearable multi-light-source fluorescence imaging system provided in embodiment 2 of the present invention;
FIG. 5 is a schematic structural diagram of a wearable multi-source fluorescence imaging system provided in embodiment 3 of the present invention;
fig. 6 is a schematic light source coupling diagram of the wearable multi-light source fluorescence imaging system provided in embodiment 3 of the present invention.
The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:
the device comprises a lens body 1, a light-emitting unit 2, a coupling optical fiber bundle 3, a dichroic mirror 4, an objective lens 5, a fluorescent light filter 6, an imaging lens 7, an image acquisition unit 8 and an acquisition control unit 9.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a wearable multi-light-source fluorescence imaging system for experimental animals, as shown in fig. 1, comprising: the lens body, the light source light path and the fluorescence imaging light path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue; the device comprises a plurality of light-emitting units, a coupling optical fiber bundle, a dichroic mirror and an objective lens, wherein the light-emitting units are used for respectively generating fluorescence excitation light or stimulation light with different wavelengths;
the light emitting unit comprises a light source and a driving module thereof, the light source is a high-power LED-narrow band filter combination or an external laser, and the driving module is used for modulating the light source according to an external TTL signal.
The coupling optical fiber bundle comprises a plurality of optical fibers, preferably plastic optical fibers, the light outlet ends of the coupling optical fiber bundle are closely arranged, and the light inlet end of the coupling optical fiber bundle is divided into a plurality of sub-bundles; the light-incoming ends of the sub-beams are respectively coupled with the light-emitting units in a matching way; the optical fibers of the plurality of sub-beams are arranged in a mixed manner in the coupling optical fiber bundle, so that the emergent light spots of the light sources from different branches at the emergent end of the optical fiber bundle are superposed, the distance from the emergent end of the coupling optical fiber bundle to the rear end face of the objective lens is within 2.5 times of the focal length, and in fact, when the distance from the emergent end of the coupling optical fiber bundle to the rear end face of the objective lens is smaller, the coupling optical fiber bundle has better emergent effect and smaller device volume, and considering the realizability, the distance from the emergent end to the rear end face of the objective lens is within 2.5 times of the focal length; the diameter of the coupling optical fiber bundle is between 0.3 and 3.0 millimeters, and the ray density of the sub-bundle is between 11 and 110 pieces per square millimeter. The light spots formed by each sub-beam have light intensity fluctuation of no more than 10% in the observation range of the visual field. The light outlet end of the coupling optical fiber bundle is wrapped by a section of rigid sleeve and is relatively fixed with the fluorescence imaging optical path; preferably, the light homogenizing device can be arranged at the emergent end of the beam combination optical fiber bundle and comprises a light homogenizing sheet or an oil coating, a light dispersion glue and a large-core-diameter coupling optical fiber.
The optical fibers from all branches are uniformly and mixedly arranged when being combined, so that the emergent light spots of the light sources from different branches at the light-emitting end of the optical fiber bundle are ensured to be superposed, and the in-situ multi-wavelength imaging or in-situ optogenetic stimulation-fluorescence imaging is realized. Meanwhile, a plurality of single optical fibers distributed at each position emit light together, so that Gaussian spots can be prevented from being formed, platform-like spots are formed, uniformity of imaging light irradiation and light genetic light irradiation is guaranteed, and a light homogenizing device can be arranged at an emergent end of a beam-combining optical fiber bundle to further increase uniformity of the spots. Under the condition that the position of the mirror body where the light outlet end is located is determined, the range of the sample illumination area can be further changed by increasing or decreasing the number of the optical fibers in the optical fiber bundle to change the diameter of the optical fiber bundle, for example, large-field imaging is realized. In addition, because the light energy is dispersed into each single optical fiber through the optical fiber bundle, the transmission power of the single optical fiber is low, and the light power with higher power can be used for optogenetic research or other researches requiring high-power light irradiation.
The fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information; the device comprises an imaging unit, an image acquisition unit, a dichroic mirror and an objective lens; the imaging unit comprises an imaging lens, preferably a fluorescent filter, the filter is preferably arranged between the dichroic mirror and the imaging lens, preferably a band-pass filter, and stray light outside a fluorescent wavelength range can be inhibited; the image acquisition unit comprises an array image detector and a peripheral circuit thereof, the array image detector is preferably a multicolor array image detector, the image acquisition unit is positioned on the rear image surface of the imaging lens, and the focal length of the imaging lens is determined by the system magnification and the effective focal length of the objective lens;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the dichroscope is preferably a multi-cut-off wavelength dichroscope, and is used for reflecting the fluorescent excitation light and/or the stimulation light with different wavelengths generated by the light emitting unit of the light source light path at the same time.
The light emitting unit of the light source light path generates fluorescence excitation light and/or stimulation light with different wavelengths, the fluorescence excitation light and/or the stimulation light are respectively coupled to corresponding sub-beams, meanwhile, the fluorescence excitation light and/or the stimulation light are transmitted to the dichroic mirror through the optical fiber beams, and the reflected fluorescence excitation light and/or the stimulation light are projected to a target area through the objective lens; the fluorescence emitted by the target area is collected by the same objective lens and is transmitted through the dichroic mirror, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
The mirror body is used for arranging the light source light path and the fluorescence imaging light path, and is made of rigid light-tight high polymer material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
The wearable multi-light source fluorescence imaging system for experimental animals provided by the invention has great challenges from two aspects: on one hand, as wearable equipment for experimental animals, the influence on the activity of the experimental animals needs to be reduced as much as possible, so that the equipment is light and free to move as much as possible; on the other hand, since the performance of the imaging system is further required to be improved and effects such as multicolor fluorescence imaging and fluorescence imaging combined with optogenetic stimulation are desired to be achieved, the light source structure is further complicated and heavy. The challenges of the two aspects form a certain contradiction, and the existing technical means are difficult to solve. The external light source is guided by the coupling optical fiber bundle, so that the weight increase of the wearable equipment caused by the complicated function of the light source is avoided, even the weight of the wearable equipment is reduced, a light outlet end of the coupling optical fiber bundle is matched with the objective lens, and a focusing lens and an optical filter of a light source light path are omitted, so that the wearable equipment is lighter, and the cost is obviously reduced; meanwhile, an external light source enters the imaging system through fiber bundle coupling, and the fiber bundle can realize switching of the collected fluorescence types (green and red) or the stimulation light wave band by replacing a light-emitting unit coupled with the fiber bundle and/or redistributing the sub-beams; the wavelength is replaced simply, the applicability is wide, and complex functions including multicolor simultaneous fluorescence imaging and in-situ optogenetic imaging (the target tissue of optogenetic stimulation laser, namely the target area of fluorescence imaging, simultaneous apposition imaging, so-called original optogenetic imaging) are realized. In order to further reduce the limitation on the free movement of the experimental animal, the invention preferably adopts the coupling optical fiber bundle of the plastic optical fiber, the plastic optical fiber has better light guiding property and flexibility, the optical fiber bundle has better flexibility and lighter weight than the plastic or quartz optical fiber with the same diameter, and particularly the optical fiber bundle is not easy to break and can carry out long-time in-vivo experiment. If the quartz optical fiber or the glass optical fiber is integrated, the whole flexibility of the coupling optical fiber bundle is poor, so that the experimental animal is not free to move or the problem of easy breakage is caused.
Under the condition that the position of the mirror body where the light-emitting end of the coupling optical fiber bundle is located is determined, the diameter of the optical fiber bundle is changed by increasing or decreasing the number of optical fibers in the optical fiber bundle, so that the range of an illumination area at the sample can be further changed and improved, for example, large-field imaging is realized. In addition, because the light energy is dispersed into each single optical fiber through the optical fiber bundle, the transmission power of the single optical fiber is low, and the light power with higher power can be used for optogenetic research or other researches requiring high-power light irradiation. Meanwhile, the number of optical wavelengths can be increased by simply increasing the number of optical fiber bundle branches, the requirements of multicolor fluorescence imaging and in-situ optogenetic-fluorescence imaging research can be met, the mirror body does not need to be modified, the size, the weight and expensive micro optical devices do not need to be additionally increased, the cost is low, the weight is light, and the method is suitable for large-scale popularization and application.
The following are examples:
example 1 in situ optogenetic-Monochromatic fluorescence imaging
A wearable multi-light source fluorescence imaging system for experimental animals, as shown in fig. 1 and 2, comprising: the lens body, the light source light path and the fluorescence imaging light path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue; the device comprises a plurality of light-emitting units, a coupling optical fiber bundle, a dichroic mirror and an objective lens, wherein the light-emitting units are used for respectively generating fluorescence excitation light or stimulation light with different wavelengths;
the light-emitting unit comprises a light source and a driving module thereof, the light source is a first external laser and a second external laser which are both provided with standard FC interfaces, the first external laser generates exciting light of 450nm, the output power can reach 50mw, the second external laser generates stimulating laser of 633nm, the output power can reach 100mw, and the driving module is used for modulating the second laser according to an external TTL signal to emit light stimulating laser according to experimental requirements.
The coupling optical fiber bundle comprises 19 plastic optical fibers, wherein light outlet ends of the plastic optical fibers are closely arranged, and light inlet ends of the plastic optical fibers are divided into two sub-bundles; the coupling is respectively connected with the light-emitting unit through a standard FC interface; the multiple optical fibers of each sub-beam are uniformly distributed at the light-emitting end and are used for forming platform-like light spots respectively, the optical fibers of the multiple sub-beams are arranged in a coupling optical fiber bundle in a mixed manner, as shown in fig. 3, the light-emitting spots of light sources from different branches at the light-emitting end of the optical fiber bundle are ensured to be superposed, and the distance from the light-emitting end of the coupling optical fiber bundle to the rear end face of the objective lens is within 2.5 times of the focal length; the coupling fiber bundle has a diameter of 0.625 mm, and the fiber density of each sub-bundle is 51 (first sub-bundle) and 46 (second sub-bundle) per square mm, respectively. The light-emitting end of the coupling optical fiber bundle is wrapped by a section of rigid sleeve and fixed at the near-dichroic mirror, and the distance between the light-emitting end and the rear end face of the objective lens is 2 times of the focal length
The coupling optical fiber bundle is provided with a light homogenizing device at the light outlet end, and the light homogenizing device is arranged at the light outlet end of the coupling optical fiber bundle so that light intensity fluctuation of light spots formed by each sub-bundle in a visual field observation range is 10%.
The light homogenizing device can be one or a combination of the following devices: 1. a light homogenizing sheet can be attached to the emergent end of the combined coupling optical fiber bundle; 2. the diameter range of the thick plastic optical fiber connected to the emergent end of the combined coupling optical fiber bundle is 0.5-3mm, and the length is 3-100 mm; 3. the sleeve at the exit end of the beam-combining optical fiber bundle can be filled with light dispersion glue.
The fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information; the device comprises an imaging unit, an image acquisition unit, an acquisition control unit, a dichroic mirror and an objective lens; the imaging unit is provided with a 500-550nm transmission band-pass fluorescence filter and an imaging lens with the diameter of 5mm and the focal length of 10mm along the light path; the image acquisition unit comprises an array image detector and a peripheral circuit thereof, wherein the array image detector is a CMOS and is connected with an upper computer through a high-speed interface, the acquisition rate can reach more than 30 frames per second, the acquisition starting time of each frame of image is output to the acquisition control unit in a pulse form for acquisition and recording, and the image acquisition unit is positioned on the rear image surface of the imaging lens; the acquisition control unit can be communicated with an upper computer, TTL pulses with fixed frequency and duty ratio are edited through software and are output to the light source module through the acquisition control unit, and modulation of the thorn laser source is achieved;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the two-phase color size is 5 × 4 × 1mm, 500-550 transmission is realized, the rest visible light wave band is reflected at 45 degrees, the diameter of the objective lens is 1.8mm, the focal length is 1.71mm, and the optical magnification of the system is 5.85 times.
The light-emitting unit of the light source circuit generates 633nm light genetic stimulation light, the light genetic stimulation light is coupled to the first sub-beam, the fluorescence excitation light with the wavelength of 450nm is coupled to the second sub-beam, meanwhile, the fluorescence excitation light is conducted to the dichroic mirror through the optical fiber bundle, and the reflected light is projected to a target area through the objective lens; the neuron activity in the target area is accompanied with the emission of fluorescence, the fluorescence is collected by the same objective lens and is transmitted through the dichroscope, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
The mirror body is used for arranging the light source light path and the fluorescence imaging light path, and is made of a polyformaldehyde resin material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
The wearable multi-light-source fluorescence imaging system provided by the embodiment can realize in-situ optogenetic stimulation-fluorescence imaging, namely, fluorescence emitted by the same part and stimulation at the same time is collected in a target region of optogenetic stimulation, and even fluorescence image information before and after stimulation can be continuously recorded, so that the condition of target neurons under direct and continuous optogenetic stimulation of the target region can be realized, and no time or space difference exists. In addition, the optical fiber bundle is soft, light and not easy to damage, and can not influence the free movement of animals.
EXAMPLE 2 two-color fluorescence imaging System
A wearable multi-light source fluorescence imaging system for experimental animals, as shown in fig. 1 and 2, comprising: the lens body, the light source light path and the fluorescence imaging light path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue; the device comprises two light-emitting units for respectively generating fluorescence excitation light with two wavelengths, a coupling optical fiber bundle, a dichroic mirror and an objective lens;
the light-emitting unit comprises a light source and a driving module thereof, the light source is a 470nm high-power LED-narrow band filter combination and a 560nm LED high-power LED-narrow band filter combination, and the driving module is used for modulating the light source according to an external TTL signal. The two light sources are lightened in a time-sharing mode in cooperation with the imaging frame frequency of the imaging unit.
The coupling optical fiber bundle comprises 37 plastic optical fibers, wherein each optical fiber is 0.125 micrometer, the light outlet ends of the optical fibers are closely arranged, and the light inlet ends of the optical fibers are divided into 2 sub-bundles as shown in fig. 2; each sub-beam is directly interfaced to the filter through the FC interface; the multiple sub-beams are uniformly distributed at the light outlet end of each optical fiber and are used for respectively forming platform light spots, the optical fibers of the multiple sub-beams are arranged in a hybrid manner in the coupling optical fiber bundle, as shown in fig. 4, the light outlet spots of the light sources from different branches at the light outlet end of the optical fiber bundle are enabled to be overlapped, and the light outlet end of the coupling optical fiber bundle is located at the rear end of the objective lens by 1.8 times of focal length; the coupling fiber bundle has a diameter of 0.875 mm, and the densities of the two sub-bundles of fibers are 43 (first sub-bundle) and 41 (second sub-bundle) per square mm, respectively. And the light outlet end of the coupling optical fiber bundle is wrapped by a section of rigid sleeve and is fixed with the fluorescence imaging light path through AB glue.
The light spot formed by each sub-beam has a light intensity fluctuation of 10% in the observation range of the visual field.
The fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information; the device comprises an imaging unit, an image acquisition unit, a dichroic mirror and an objective lens; the imaging unit is provided with a 500-550nm,580-620nm double band-pass filter and an imaging lens with the diameter of 5mm and the focal length of 10mm along the light path; the image acquisition unit comprises an array image detector and a peripheral circuit thereof, the array image detector is a 480 x 752 gray array image detector, and the image acquisition unit is positioned on the rear image surface of the imaging lens;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the two-phase color is transmitted at 550nm,580 nm and 620nm, and the rest visible light wave band is reflected at 45 degrees; the diameter of the objective lens is 1.8mm, the focal length is 1.71mm, and the optical magnification of the system is 5.85 times
The light-emitting unit of the light source light path generates 470 fluorescent excitation light which is coupled to the first sub-beam, generates 560nm fluorescent excitation light which is coupled to the second sub-beam, is transmitted to the dichroic mirror by the optical fiber beam, and is projected to a target area through the objective lens after being reflected; the neuron activity in the target area is accompanied with the emission of fluorescence, the fluorescence is collected by the same objective lens and is transmitted through the dichroscope, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
The mirror body is used for arranging the light source light path and the fluorescence imaging light path, and is made of rigid light-tight high polymer material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
The wearable multi-light-source fluorescence imaging system provided by the embodiment can realize in-situ bicolor fluorescence imaging, namely, in a target area, fluorescence emitted by neurons marked by two kinds of fluorescence is collected, the activity of the neurons of different types can be continuously observed at the same time, and richer neural loop information can be acquired. Simple structure, the optic fibre bundle is soft, light, not fragile, can not exert an influence to animal free activity. The light source expansion can be conveniently carried out.
Example 3 in situ optogenetic-two-color fluorescence imaging
A wearable multi-light source fluorescence imaging system for experimental animals, as shown in fig. 2, 5, comprising: the lens body, the light source light path and the fluorescence imaging light path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue; the device comprises a plurality of light-emitting units, a coupling optical fiber bundle, a dichroic mirror and an objective lens, wherein the light-emitting units are used for respectively generating fluorescence excitation light or stimulation light with different wavelengths;
the light-emitting unit comprises a light source and a driving module thereof, the light source is a 470nm high-power LED-narrow band filter combination, a 560nm high-power LED-narrow band filter combination and a 633nm external laser, and the driving module is used for modulating the light source according to an external TTL signal. The-470 nmLED light source and the-560 nmLED light source are double-color fluorescence excitation light sources, and are matched with the imaging frame frequency of the imaging unit, and the two light sources are lightened in a time-sharing manner to realize double-color fluorescence imaging; the 633nm light source is an optogenetic light stimulating laser source and is modulated by a TTL signal with fixed frequency and duty ratio. The TTL pulse signal comes from the acquisition control unit.
The coupling optical fiber bundle comprises 37 plastic optical fibers, wherein light outlet ends of the plastic optical fibers are closely arranged, and light inlet ends of the plastic optical fibers are divided into 3 sub-bundles; the plurality of sub-beams are uniformly distributed at the light outlet end of each optical fiber and are used for respectively forming platform light spots, the optical fibers of the plurality of sub-beams are arranged in a coupling optical fiber bundle in a mixed manner, as shown in fig. 6, the light outlet spots of light sources from different branches at the light outlet end of the optical fiber bundle are enabled to be overlapped, and the light outlet end of the coupling optical fiber bundle is positioned at the rear end of the objective lens by 2 times of focal distance; the coupling fiber bundle has a diameter of 0.875 mm, and the fiber densities of the sub-bundles are 46 (first sub-bundle), 42 (second sub-bundle) and 42 (third sub-bundle) per square mm, respectively. The light-emitting end of the coupling optical fiber bundle is wrapped by a section of rigid sleeve and is fixed with the fluorescence imaging light path through quick-drying glue
The light-emitting end of the coupling optical fiber bundle is provided with a light-homogenizing device, the light-emitting end of the coupling optical fiber bundle is continuously connected with a plastic optical fiber bundle with the diameter of 1mm and the length of 30mm, and light spots formed by each sub-bundle have light intensity fluctuation of 10% in the visual field observation range.
The fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information; the device comprises an imaging unit, an image acquisition unit, a dichroic mirror and an objective lens; the imaging unit is provided with a 500-550nm,580-620nm double-band-pass fluorescence filter and an imaging lens with the diameter of 5mm and the focal length of 12.5mm along the light path; the image acquisition unit comprises an array image detector and a peripheral circuit thereof, the array image detector is a 480 x 752 gray array image detector, and the image acquisition unit is positioned on the rear image surface of the imaging lens;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the two-phase color is transmitted at 550nm,580-620nm, the rest visible light wave band is reflected at 45 degrees, the diameter of the objective lens is 1.8mm, the focal length is 1.71mm, and the system magnification is 7.31 times.
The light-emitting unit of the light source light path generates 633nm light genetic stimulation light to be coupled to the first sub-beam, generates 560mn fluorescence excitation light to be coupled to the second sub-beam, generates 470nm fluorescence excitation light to be coupled to the third sub-beam, is conducted to the dichroic mirror by the optical fiber beam, and is projected to a target area through the objective lens after being reflected; the neuron activity in the target area is accompanied with the emission of fluorescence, the fluorescence emitted by the target area is collected by the same objective lens and is transmitted through the dichroscope, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
The mirror body is used for arranging the light source light path and the fluorescence imaging light path, and is made of rigid light-tight high polymer material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
The wearable multi-light-source fluorescence imaging system provided by the embodiment can realize in-situ two-color fluorescence imaging and optogenetic stimulation, namely, in a target area, fluorescence emitted by neurons of two fluorescence labels is collected, the activity of the neurons of different types can be continuously observed at the same time, the activity of the neurons can be regulated and controlled at the same time, and even fluorescence image information before and after stimulation can be continuously recorded. Simple structure, the optic fibre bundle is soft, light, not fragile, can not exert an influence to animal free activity. The light source expansion can be conveniently carried out.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A fluorescence imaging system for experimental animals is characterized by comprising a light source light path and a fluorescence imaging light path;
the light source circuit is used for projecting a light source on the target fluorescence labeling tissue;
the fluorescence imaging optical path is used for collecting fluorescence signals emitted by a target fluorescence labeling tissue and forming fluorescence image information;
the light source light path and the fluorescence imaging light path share a dichroic mirror and an objective lens; the light source circuit also comprises a coupling optical fiber bundle and a plurality of light emitting units for respectively generating fluorescence excitation light or optogenetic stimulation light with different wavelengths; the coupling optical fiber bundle comprises a plurality of optical fibers, wherein the light outlet ends of the optical fibers are closely arranged, and the light inlet ends of the optical fibers are divided into a plurality of sub-beams; the light-incoming ends of the sub-beams are respectively coupled with the light-emitting units in a matching way; the optical fibers of the plurality of sub-beams are arranged in the coupling optical fiber bundle in a mixed manner, and the distance from the light outlet end to the rear end face of the objective lens is within 2.5 times of the focal length; the fluorescence imaging light path further comprises an imaging unit and an image acquisition unit;
the light emitting unit of the light source light path generates fluorescence excitation light and/or stimulation light with different wavelengths, the fluorescence excitation light and/or the stimulation light are respectively coupled to corresponding sub-beams, meanwhile, the fluorescence excitation light and/or the stimulation light are transmitted to the dichroic mirror through the optical fiber beams, and the reflected fluorescence excitation light and/or the stimulation light are projected to a target area through the objective lens; the fluorescence emitted by the target area is collected by the same objective lens and is transmitted through the dichroic mirror, and the fluorescence is imaged to the image acquisition unit by the imaging lens to form fluorescence image information.
2. The fluorescence imaging system for experimental animals according to claim 1, wherein said coupling fiber bundle has a diameter of 0.3-3.0 mm and a sub-bundle fiber density of 11-110 fibers/mm.
3. The fluorescence imaging system for experimental animals according to claim 1, wherein the light intensity fluctuation in the observation range of the field of view of the light spot formed by each sub-beam of the coupled optical fiber bundle is not more than 10%.
4. The fluorescence imaging system for experimental animals as claimed in claim 1, wherein the light exit end of the coupling fiber bundle is wrapped by a section of rigid sleeve and fixed relative to the fluorescence imaging optical path.
5. The fluorescence imaging system for experimental animals as claimed in claim 1, wherein the light-exiting end of the coupling fiber bundle is provided with a light-homogenizing device, and the light-homogenizing device comprises a light-homogenizing sheet, light dispersion glue and a large-core coupling fiber.
6. The fluorescence imaging system for experimental animals according to claim 1, wherein said optical fiber is a plastic optical fiber.
7. The fluorescence imaging system for experimental animals as claimed in claim 1, wherein said light emitting unit comprises a light source and a driving module thereof, said light source is a high power LED-narrowband filter combination or an external laser; preferably, the driving module is configured to modulate the light source according to an external TTL signal.
8. The fluorescence imaging system for experimental animals according to claim 1, wherein the dichroic mirror is a multi-cut-off wavelength dichroic mirror for reflecting the fluorescence excitation light and/or the excitation light of different wavelengths generated by the light emitting unit of the light source path at the same time.
9. The fluorescence imaging system for experimental animals according to claim 1, wherein the imaging unit of the fluorescence imaging optical path comprises an imaging lens, preferably a fluorescence filter, and the filter is preferably between the dichroic mirror and the imaging lens; the image acquisition unit of the fluorescence imaging light path comprises an array image detector and a peripheral circuit thereof, and the array image detector is preferably a multicolor array image detector.
10. The fluorescence imaging system for experimental animals as claimed in claim 1, wherein said fluorescence imaging system comprises a mirror body for setting said light source optical path and fluorescence imaging optical path, said mirror body is made of rigid opaque high molecular material; the optical fiber is fixedly packaged with the light-emitting end of the coupling optical fiber bundle.
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