CN106706585A - Device and method for recording living body fluorescence signals - Google Patents

Abstract

The invention discloses a device and method for recording living body fluorescence signals. The device comprises a light source and a detection device. The light source comprises a laser transmitter and a transmission optical fiber. The tail end of the transmission optical fiber is connected with an insertion optical fiber, the insertion optical fiber is a light beam with a casing wrapped with stainless steel, part of optical fiber is used for exciting light projection, and the remaining optical fiber is used for sampling and collecting the fluorescence signals. The detection device comprises a light filtering device (an optical filter group), a spectrometer and a transmission optical fiber. Monkey brain viruses can be monitored in real time through the fluorescence signals, experimental efficiency is improved, and manpower and material resources are saved. In addition, the device is simple to operate and low in cost and can detect deep fluorescence signals in brains of different living body animals.

Description

A device and method for recording fluorescent signals in a living body

technical field

The invention belongs to the central nervous system living body fluorescent signal recording in the technical field of biological visible light imaging, and in particular relates to a living body fluorescent signal recording device and method.

Background technique

With the rise of gene technology, a series of emerging technologies such as optogenetics, retrovirus tracer, gene knockout and gene therapy play an important role in neuroscience research. The realization of all these technologies depends on the injection of different carrier viruses into the target organs, and whether these viruses can successfully transfect the target cells often becomes the key to the success or failure of the experimental operation. Usually, we use fluorescent signals to detect whether the virus has transfected the target cells, whether it is working normally in the experimental animal body, and the extent of the infected tissue. In the past fluorescent signal detection, it is usually necessary to sacrifice animals to verify the obtained tissue samples, and then after a series of fixation, dehydration and sectioning, the prepared tissue section slides are placed under a fluorescent microscope for observation one by one. This is not only time-consuming, labor-intensive and expensive (in the case of advanced animal experiments such as rhesus monkeys), especially in the field of neurological research, researchers are often required to continue to observe the behavior of the same experimental animal or other measurement indicators after completing the virus transfection operation, or to study What people often need to observe is the continuous change of a certain process. In the past, researchers generally performed the same treatment on a batch of animals, sacrificing animals at different periods of time to obtain specimens. Such a method is not only costly, time-consuming and labor-intensive, but also cannot avoid the factors of individual differences among individuals. In this way, animals cannot be killed in advance to detect whether the virus transfection is successful, and all experiments can only be performed after completing all experiments. This will waste a lot of increased workload and reduce experimental efficiency.

In the actual scientific research process, especially in primate experiments, the precious primate resources determine that this is a difficult task to complete. In transgenic experiments using viruses as carriers, how to observe fluorescent signals in vivo, especially in vivo fluorescent signals in deep brain tissue, and then observe the expression of viruses in experimental animals and the expression of transgenes, It is of great significance to control the quality of the transgenic process and improve the success rate of later experiments.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a device and method for continuously observing fluorescent signals in a living body, especially a recording device and method for fluorescent signals in a living body.

The technical solution of the present invention: a recording device and method for fluorescent signals in living body, including two parts, an excitation light source and a detection device. First, insert the animal brain tissue into a set of precision-processed optical fiber bundles, one of which is used to connect the laser to transmit excitation light, and the other set of optical fibers is used to receive and transmit fluorescent signals in the brain, and the excited fluorescence passes through this set of fibers at the same time. The optical fiber is sent out, the background light signal is filtered by the filter device, and then enters the spectrometer, and the real-time fluorescent signal can be recorded in the brain of the living animal. Through the optical fiber bundle inserted into the animal's brain tissue, one of the optical fibers is used to connect the laser to transmit the excitation light, and the other group of optical fibers is used to receive and transmit the fluorescent signal in the brain, and the fluorescence generated by the excitation is transmitted through this group of optical fibers at the same time and filtered After the optical device filters out the background light signal and enters the spectrometer, the real-time fluorescent signal can be recorded in the brain of the living animal.

The cannula is implanted in the animal brain in advance, and then the fiber bundle is inserted into the cannula and connected to the laser emitter and spectrometer, and the fluorescent signal can be detected in the living brain, and the change of the strength of the fluorescent signal can be observed in real time (can be used for Detection of functional protein fluorescence such as Gcamp).

The present invention has the following salient features:

1. Observe the fluorescent signal without sacrificing experimental animals. The light source part and the fluorescent signal recording part of the device are two separate and adjustable independent devices. Take advantage of the portability and flexibility of this device without sacrificing the animal for tissue sectioning.

2. Wide range of applications. For different fluorescent proteins, researchers only need to change different filters in the light source part to obtain excitation light of different wavelengths, and also change filters of specific wavelengths in the recording part to realize the recording of fluorescence signals of different fluorescent proteins. In addition, by selecting optical fibers with different diameters, the irradiation range of the light source can be controlled, so that fluorescent signal recording in various ranges can be realized.

3. The cost is lower than most existing fluorescent signal recording devices. The device mainly includes an excitation light source, a filter, an optical fiber, and a spectrometer.

4. It can realize the collection of fluorescent signals in vivo, especially the in vivo collection of fluorescent signals from deep intracranial tissues of large animals.

Description of drawings

Fig. 1 is the schematic diagram of the principle of the recording device of the fluorescent signal of the living body according to the present invention;

Fig. 2 is a schematic diagram of the structure of an optical fiber inserted into animal brain tissue using the present invention.

detailed description

As shown in FIG. 1 , the recording device for the fluorescent signal of the living body described in this embodiment includes two parts: a light source and a detection device. The light source part includes a laser transmitter and a transmission fiber. The end of the transmission fiber is connected to the insertion fiber. The insertion fiber is a light bundle wrapped in stainless steel. Some of the fibers are used for the projection of excitation light, and the rest are used for sampling and collecting fluorescence. Signal. The detection device includes a filter device (filter group), a spectrometer and transmission fiber.

The optical filter device used in the present invention includes a light shield, and an optical filter stuck on the optical filter groove, connected to the SMA interface of the device, and the optical filter can be replaced at any time. Both ends of the device are connected with optical fibers. After the fluorescence emitted from the monkey brain passes through the device, background light signals other than the target fluorescence can be filtered out. At the same time, a filter device can also be connected to the laser end to generate laser light with a specific wavelength.

During the specific implementation process, a cannula matching the insertion optical fiber (structure shown in Figure 2) was embedded in the macaque brain through surgery in advance to the vicinity of the observation target area, and the insertion optical fiber was inserted through the cannula into the area to be observed in the monkey brain. The excitation light is introduced into the area to be observed in the monkey brain through the transmission optical fiber and the insertion optical fiber. The fluorescent signal generated by excitation passes through the optical fiber and filter device (filter set) and then enters the spectrometer through the optical fiber. Part of the excitation light projected into the monkey brain is also transmitted through the optical fiber after being reflected, but it is blocked when it passes through the optical filter. , so only the fluorescence generated after excitation enters the spectrometer, and the fluorescence signal can be known by analyzing the signal of the spectrometer.

The schematic diagram of the optical fiber inserted into the monkey brain tissue is shown in Figure 2. The optical fiber is composed of 6 optical fiber bundles with the same core diameter. Two groups of optical fiber bundles 12, 13 in the figure, one group is used to transmit the excitation light, the other group is used to receive the excited fluorescence, the metal sleeve 11 outside the optical fiber is used to protect the optical fiber. The depth of the optical fiber inserted into the monkey brain is adjustable. Several metal rings (not shown in the figure) are set on the optical fiber metal sleeve. The thickness of each small metal ring is 0.5mm. By controlling the number of metal rings on the metal sleeve, The insertion depth of the fiber can be adjusted.

In use, the cannula is embedded in the monkey brain, and the optical fiber is inserted from the cannula into the monkey brain to maintain accuracy. There is a rubber tube and a tube cover on the periphery of the casing. After the measurement is completed, the tube cover can be covered for protection. In addition, the pluggability of the optical fiber allows multiple repeated measurements of fluorescence, so as to understand the expression of fluorescence in real time.

The present invention provides a way to simply detect fluorescent signals on a living body through the combination of a laser, an optical fiber and a filter group, and the detection depth can be adjusted and can be measured repeatedly. It is of great significance to control the quality of the transgenic process and improve the success rate of later experiments.

The above description is a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be It is regarded as the protection scope of the present invention.

Claims (7)

1. a kind of tape deck of vivo fluorescence signal, it is characterised in that：Including light source and detection means two parts, the light source Part include generating laser and Transmission Fibers, Transmission Fibers end connection insertion optical fiber, this insertion optical fiber be a shell by The light shafts of stainless steel parcel, which part optical fiber is used for the projection of exciting light, and remaining optical fiber is used for sample collection fluorescence signal； Detection means includes the filtering apparatus, spectrometer and the Transmission Fibers that are sequentially connected.

2. the tape deck of vivo fluorescence signal according to claim 1, it is characterised in that：The filtering apparatus includes lucifuge Cover, and the optical filter on optical filtering film trap is stuck in, connect the SMA interfaces of the device.

3. the tape deck of vivo fluorescence signal according to claim 1, it is characterised in that：The insertion optical fiber is by 6 phases Constituted with the fibre bundle of core diameter, one of which is used to transmit exciting light, and another set is used to receive the fluorescence for ejecting.

4. the tape deck of vivo fluorescence signal according to claim 3, it is characterised in that：Set outside the insertion optical fiber There is metal sleeve, for protecting optical fiber.

5. the tape deck of vivo fluorescence signal according to claim 4, it is characterised in that：If being set on the metal sleeve Dry quoit, each metal ringlet thickness is 0.5mm, adjustable by controlling to increase the quoit quantity on metal sleeve Save the insertion depth of optical fiber.

6. a kind of recording method of vivo fluorescence signal, it is characterised in that：Insertion animal brain is one group of fibre bundle, wherein one Root optical fiber is used for connecting laser and transmits exciting light, and another group of optical fiber is used for receiving and transmitting the fluorescence signal of intracerebral, excites product Raw fluorescence is spread out of by this group of optical fiber simultaneously, by entering spectrometer after filtering apparatus wiping out background optical signal, just can be in work Real-time fluorescence signal is recorded in body animal brain.

7. the recording method of vivo fluorescence signal according to claim 6, it is characterised in that：The prior heeling-in set in animal brain Pipe, afterwards by the fibre bundle plug-in-sleeve and connect generating laser and spectrometer and can live body intracerebral detect fluorescence letter Number.