CN114869236A - In-vivo animal biophoton imaging detection system and detection method - Google Patents

Abstract

The invention relates to a system and a method for detecting in-vivo animal brain biophoton imaging, wherein the system comprises an ultra-weak biophoton imaging system UBIS, a movable box, an optical fiber bundle and an optical fiber bundle fixing sleeve device; the optical fiber bundle fixing sleeve device is fixed on the skull of an animal to be tested, one end of the optical fiber bundle is fixed below a photon imaging device in a UBIS dark box of the ultra-weak biological photon imaging system, and the other end of the optical fiber bundle is connected with the optical fiber bundle fixing sleeve device which is placed on the skull of the animal to be tested in the movable box. The animal firstly opens the skull and exposes the cortical area, one end of the optical fiber bundle is connected with the optical fiber bundle sleeve device fixed on the skull of the animal to be detected and is used for transmitting the biophoton activity of the cortical area exposed under the sleeve; the other end is fixed under an EM-CCD lens in a dark box, and the biophotonic activity of the cortex area is detected in real time. The invention uses the optical fiber bundle to transmit the biophoton activity, thereby achieving the real-time detection of the biophoton activity state of the cerebral cortex of the in vivo animal.

Description

In-vivo animal biophoton imaging detection system and detection method

Technical Field

The invention relates to a cerebral cortex biological photon imaging detection system and a detection method which are constructed by utilizing the capacity of transmitting ultra-weak photons through an optical fiber bundle. The invention discloses an in-vivo animal cerebral cortex biophoton detection system, which is applied to in-vivo biophoton imaging and relates to the fields of neuroscience, biomedicine, photonics, medical imaging and the like.

Background

The activity and transmission of biophotons on the neural circuits are supported experimentally and theoretically, so far, experimental evidence mainly comes from the detection of the neurobiophotons by using an ex vivo brain slice, and in addition, few literature reports on the neural signal coding mechanism of the biophotons exist, and a Russian scholarly proposes the idea of intensity and frequency coding, but lacks detailed mathematical modeling and experimental confirmation. The successful implementation of quantum communication in recent years suggests that the mechanism of neural signal encoding for biophotonic molecules may be similar to that of quantum communication. Compared with electrical signals and chemical signals, the biological photons used as information transmission, processing and coding media have distinct advantages, including high transmission speed, large information carrying capacity, low energy consumption and the like. The phenomenon that the spectrum of the biological photon activity and transmission induced by the glutamic acid appears to be red-shifted from animals (bullfrog, mice, chicken, pigs and macaque in turn) to human beings is found, and the phenomenon provides an important basis for explaining the biological photon information transmission and coding mechanism. Therefore, the information transmission and coding mechanism model of the brain is constructed based on the biophoton information transmission mechanism, and has important theoretical significance and application value. We performed craniotomy on anesthetized mice in the early stage and placed them in a biophotonic imaging system with the exposed cerebral cortex facing the EM-CCD lens. And (3) imaging the cortical region in real time by adopting an ultra-weak biophotonic imaging system. Image information is extracted and analyzed using image analysis software. The experimental result shows that the exposed cerebral cortex has obvious biological photon radiation, which indicates that biological photon activity can be detected through the cerebral cortex. The method utilizes different paradigm visual and auditory stimuli to stimulate animals and recent smell and olfaction stimuli to detect that the photon radiation of the brain cortex of the in vivo animal presents different intensity change characteristics, and initially constructs an in vivo animal brain cortex biophotonic imaging system. On the basis of the technology, the method can be applied to detecting the photon radiation change characteristics of animal cerebral cortex induced by other stimulation modes. Provides a research method means for deeper understanding of the 'photon brain' mechanism.

Disclosure of Invention

The invention aims to provide an in-vivo animal brain biophoton imaging detection system and a detection method for detecting in-vivo animal brain cortical area biophoton radiation characteristics.

The technical scheme of the invention is as follows:

an in vivo animal brain biological photon imaging detection system comprises an ultra-weak biological photon imaging system UBIS, a movable box, an optical fiber bundle and an optical fiber bundle fixing sleeve device; the method is characterized in that: the optical fiber bundle fixing sleeve device is fixed on the skull of an animal to be tested, one end of the optical fiber bundle is fixed below a photon imaging device in a UBIS dark box of the ultra-weak biological photon imaging system, and the other end of the optical fiber bundle is connected with the optical fiber fixing sleeve device placed on the skull of the animal to be tested in the movable box.

The invention also comprises a stimulation device which is arranged in the movable box and is used for stimulating the animal to be tested.

The stimulation device is a Bluetooth intelligent device and can perform stimulation including light, sound, smell, electricity, heat and/or medicines.

The optical fiber bundle fixing sleeve device is composed of an insertion pipe, a sleeve and a nut, wherein one end of the insertion pipe is provided with a platform, one end of the sleeve is provided with an external thread, the nut is provided with an internal thread, one end of the optical fiber bundle is connected with the insertion pipe, the insertion pipe penetrates through the nut and the platform to be clamped in the nut, the nut and the sleeve are tightly fixed through the thread, the other end of the sleeve is fixed on the skull of an animal to be detected, and the bottom of the sleeve is an exposed cerebral cortex area of the animal to be detected.

The optical fiber bundle is formed by bonding more than 9 optical fibers into a bundle by using an adhesive, and two ends of the optical fiber bundle are respectively embedded with a ceramic ferrule; all optical fibers are sleeved in a black protective sleeve, and the two ends of the protective sleeve are bonded with the metal tail handle of the ceramic ferrule and are wrapped and shielded from light.

The optical fiber is a multimode or single-mode optical fiber.

The brain biophoton is ultra-weak biophoton which is autonomously radiated by the brain.

One end of the optical fiber bundle is fixed above a cerebral cortex area exposed by an animal to be detected, the other end of the optical fiber bundle is fixed below an EM-CCD lens, the cortical biophoton activity of the animal to be detected is induced by a stimulation device, the optical fiber bundle transmits the cortical biophoton activity of the animal to be detected, and the EM-CCD detects the radiation state of biophoton. The invention adopts an ultra-weak biological photon imaging system, patent No. 201310524951.4, the ultra-weak biological photon imaging system converts biological photon signals into electric signals, displays the biological photon activity state in real time in the form of images, and finally reflects the biological photon activity in the form of gray values, including intensity and spectrum.

The detection method using the in-vivo animal biophoton imaging detection system is characterized by comprising the following steps of:

(1) the experimental preparation operation of the ultra-weak biophoton imaging system comprises the following steps: opening an indoor air conditioner, a dehumidifier and a low-temperature cooling circulating pump, setting the temperature to be 25 ℃ and the humidity to be 40-50%, and starting an EM-CCD lens; opening a computer, starting the matched operating software of the EM-CCD, setting relevant parameters according to experiment requirements, cooling the EM-CCD system to-90 ℃ and keeping the temperature constant, and ensuring the normal operation of the system; finally, the optical fiber bundle is integrally arranged in an EM-CCD dark box, one end of the optical fiber bundle is arranged below an EM-CCD lens and is adjusted to the center of the lens and is fixed, and the other end of the optical fiber bundle is connected with an optical fiber fixing sleeve device arranged on the skull of the animal to be detected in the movable box;

(2) shooting a positioning photo: turning off all indoor light sources to ensure that the experimental environment is a dark environment; setting the EM-CCD mode as a Normal mode, setting the exposure time as 1s, clicking a lens Live mode, opening a camera bellows door to finely adjust an EM-CCD camera lens until the best shooting effect can be obtained, and finally shooting a positioning photo;

(3) collecting biological photons: closing the Live mode, tightly closing a dark box door, and turning off all indoor light sources to ensure that the experiment is carried out under the dark condition; converting the EM-CCD mode into an electron multiplication mode, setting the gain level to be 1200, and setting exposure time and the number of collected pictures corresponding to the experiment requirements;

(4) and (3) data storage: after the experiment is finished, the experimental data is stored to a relevant path document according to a certain data format; restoring the parameter setting of the EM-CCD and closing the system and other experimental instruments;

(5) extracting an average gray value: performing bright spot removal processing on the sequence diagram by using a well-written program on a software platform (such as Matlab 7.0) to avoid the influence of cosmic rays on the extraction of the gray value of the image; opening the sequence image by using EM-CCD matched software, importing a positioning photo, selecting a target area by using a software framing tool, and then taking the selected area except the target area as a background area; finally, extracting the average gray values of the target area and the background area by using a gray value extraction function, and exporting data to a data file (such as Excel) for storage;

(6) and (3) data analysis: and (5) performing data analysis and statistical analysis on the data file exported in the step (5) by using data analysis software.

The system for imaging and detecting the biological photons of the brain of the body animal is applied to the biological photon activity characteristics of the body animal.

The invention uses the optical fiber bundle to transmit the biological photon activity, thereby achieving the real-time detection of the biological photon activity state of the cerebral cortex of the in vivo animal.

Drawings

FIG. 1 is a schematic diagram of the in-vivo animal biophotonic imaging detection system of the present invention.

Fig. 2 is a disassembled schematic view of the optical fiber bundle fixing sleeve device of the present invention.

Figure 3 is a schematic representation of a representative rat odor (vanillin) stimulation experimental protocol.

Figure 4a is a plot of the course of a representative rat odor stimulation experiment.

Figure 4b is an image of a 25 th frame of a representative rat scent challenge experimental procedure.

Figure 4c is an image of a representative rat scent challenge experimental procedure at frame 35.

Fig. 4d is an image of a 45 th frame of a representative rat scent challenge experimental procedure.

Fig. 4e is an image of a representative frame 55 of the rat scent challenge experimental procedure.

Fig. 4f is an image of a representative rat scent challenge experimental procedure at frame 65.

FIG. 5a is a graph showing the variation of photon radiation activity during a representative rat odor stimulation experiment.

Fig. 5b is a graph of the mean change in photon radiation activity over the course of the distilled water control (upper line) and odor stimulation (lower line) experiments (distilled water n-10, vanillin n-8) with arrows representing the odor exposure time points for the experimental groups.

Fig. 5c is a line graph of the data of fig. 5b taken for each 15 averages.

FIG. 5d is a comparative histogram of distilled water before and after control stimulation.

Fig. 5e is a comparison histogram before and after odor challenge.

Figure 5f is a graph of a statistical analysis of the radiation activity of the motor cortex photons of distilled water control and odor stimuli over the same time period.

Detailed Description

The present invention is described below with reference to the accompanying drawings.

As shown in figure 1, the in vivo animal biophoton imaging detection system comprises an ultra-weak biophoton imaging system UBIS (only a dark box and a photon imaging device are shown in the figure), a movable box 4, an optical fiber bundle 3, a stimulation device 6 and an optical fiber bundle fixing sleeve device 5, wherein the optical fiber bundle fixing sleeve device 5 consists of an insertion tube 5-1, a sleeve 5-2 and a screw cap 5-3, one end of the optical fiber bundle 3 is fixed below the photon imaging device 1 in the ultra-weak biophoton imaging system UBIS dark box 2, the photon imaging device 1 is an EM-CCD camera and a lens, one end of the insertion tube 5-1 is provided with a platform, one end of the sleeve 5-2 is provided with an external thread, the screw cap 5-3 is provided with an internal thread, the platform of the insertion tube 5-1 has an external diameter of 11mm, an internal diameter of 5mm, an external diameter of 8mm and a thickness of 1 mm; the thickness of the cap peak of the screw cap 5-3 is 0.95mm, the inner diameter is 8.02mm, and the outer diameter is 12 mm; the outer diameter of the base of the sleeve 5-2 is 14mm, the thickness is 1mm, the inner diameter is 8.05mm, and the outer diameter is 11 mm; as shown in fig. 2, one end of the optical fiber bundle 3 is connected with the insertion tube 5-1, the insertion tube 5-1 passes through the nut 5-3 and the platform is clamped in the nut 5-3, the nut 5-3 is tightly fixed with the sleeve 5-2 through threads, the other end of the sleeve 5-2 is fixed on the skull of the animal to be tested, and the bottom of the sleeve 5-2 is an exposed cerebral cortex area of the animal to be tested; the stimulation device 6 is arranged in the movable box 4, the stimulation device 6 is a Bluetooth intelligent device, can be stimulated by light, sound, smell, electricity, heat and/or medicines and is used for stimulating the animal to be tested.

The optical fiber bundle 3 is formed by 20 multimode optical fibers which are bonded into a bundle by adhesive, the length of the optical fiber bundle is 2.5m, and two ends of the optical fiber bundle are respectively embedded with a ceramic ferrule; all optical fibers are sleeved in a black protective sleeve, and the two ends of the protective sleeve are bonded with the metal tail handle of the ceramic ferrule and are wrapped and shielded from light.

The present invention adopts an ultra-weak biophotonic imaging system patent No. 201310524951.4.

The detection method using the in-vivo animal biophoton imaging detection system is characterized by comprising the following steps of:

(1) the experimental preparation operation of the ultra-weak biophoton imaging system comprises the following steps: opening an indoor air conditioner, a dehumidifier and a low-temperature cooling circulating pump, setting the temperature to be 25 ℃ and the humidity to be 40-50%, and starting an EM-CCD lens; opening a computer, starting the matched operating software of the EM-CCD, setting relevant parameters according to experiment requirements, cooling the EM-CCD system to-90 ℃ and keeping the temperature constant, and ensuring the normal operation of the system; finally, the optical fiber bundle is integrally arranged in an EM-CCD dark box, one end of the optical fiber bundle is arranged under an EM-CCD lens and is adjusted to the center of the lens and is fixed, and the other end of the optical fiber bundle is connected with an optical fiber fixing sleeve device which is arranged on the skull of the animal to be detected in the movable box;

(2) shooting a positioning photo: turning off all indoor light sources to ensure that the experimental environment is a dark environment; setting the EM-CCD mode as a Normal mode, setting the exposure time as 1s, clicking a lens Live mode, opening a camera bellows door to finely adjust an EM-CCD camera lens until the best shooting effect can be obtained, and finally shooting a positioning photo;

(3) collecting biological photons: closing the Live mode, tightly closing a dark box door, and turning off all indoor light sources to ensure that the experiment is carried out under the dark condition; converting the EM-CCD mode into an electron multiplication mode, setting the gain level to be 1200, and setting exposure time and the number of collected pictures corresponding to the experiment requirements;

(4) and (3) data storage: after the experiment is finished, the experimental data is stored to a relevant path document according to a certain data format; restoring the parameter setting of the EM-CCD and closing the system and other experimental instruments;

(5) extracting an average gray value: performing bright spot removal processing on the sequence diagram by using a well-written program on a software platform (such as Matlab 7.0) to avoid the influence of cosmic rays on the extraction of the gray value of the image; opening the sequence image by using EM-CCD matched software, importing a positioning picture, selecting a target area by using a software framing tool, and then taking the selected area except the target area as a background area; finally, extracting the average gray values of the target area and the background area by using a gray value extraction function, and exporting data to a data file (such as Excel) for storage;

(6) and (3) data analysis: data analysis and statistical analysis were performed using data analysis software.

The specific experimental steps of the invention are as follows:

the experimental animals selected for the experiment were male 6-8 week Wister rats purchased from the experimental animals public service center (wuhan, china) in north of huh. The breeding environment is as follows: the food and water are freely taken by 12 hours of circadian rhythm illumination at room temperature of 20-25 ℃ and humidity of 30-35%. The fiber bundle was jacketed in a black protective jacket. And finally, the two ends of the high-temperature black protective sleeve are quickly bonded with the metal tail handle of the ceramic ferrule, and the metal tail handle is sealed and wound by using a black adhesive tape to avoid the influence of external light. This produces a bundle of optical fibers that transmit the biophotons.

The rats were subjected to craniotomy to fully expose the cerebral cortical areas of the rats to be examined. The center of the cannula was placed against the exposed cerebral cortical area of the rat, the cannula was fixed to the rat skull with dental cement and the rat skin was sutured. After the dental cement is solidified and the sleeve is completely fixed, inserting the cannula into the sleeve; thereby fixing the optical fiber bundle on the head of the rat; the other end of the optical fiber bundle is right opposite to the EM-CCD lens for real-time biophotonic imaging.

Vanillin odor stimulation in vivo rat cerebral motor cortex biophotonic assay

Experimental materials:

1. laboratory animal

The experimental animal is a 7-10 week old adult Wister rat with the weight of 250-300 g and the SPF level, which is purchased from the research center of experimental animals in Hubei province. A breeding environment: the temperature is 18-25 ℃, the humidity is 40-50%, the day and night rhythm illumination is 12h, rats freely eat and drink water, and the feed for feeding is common feed. For the protection of experimental animals, the damage to the animals should be minimized or reduced during the whole experimental process.

2. Solution preparation

(1) Physiological saline: 0.9g of sodium chloride was weighed out and dissolved in 100mL of ultrapure water, and the solution was stirred to be sufficiently dissolved.

(2) 10% chloral hydrate: 5g of chloral hydrate is weighed and dissolved in a proper amount of normal saline, and after the chloral hydrate is fully dissolved, the normal saline is added to the normal saline to have a constant volume of 50 mL. The solution is easy to decompose under heating and ultraviolet irradiation, and is sealed and stored in a refrigerator at 4 ℃ by using a light-resistant reagent bottle, and the shelf life of the chloral hydrate solution is 3 months.

(3) 8% of hydrogen peroxide: 13.3mL of 30% hydrogen peroxide solution is weighed, added into 36.7mL of ultrapure water, stirred evenly and stored in a refrigerator at 4 ℃.

3. Preparation of odor sources

10% vanillin: 10g of vanillin is weighed into a beaker containing 100mL of distilled water, and the beaker is placed on a magnetic stirrer to be heated and stirred until the vanillin is completely dissolved. After the solution is completely dissolved, the solution is placed in a reagent bottle for storage at normal temperature.

Experimental methods and procedures:

1. preparation before experiment

(1) The rats before operation are fasted and water is not allowed for 12 h.

(2) The high-pressure steam sterilization pot is used for sterilizing surgical instruments such as surgical forceps, precision surgical scissors, hemostatic forceps, suture curved needles and the like. And opening the air conditioner of the room, setting the temperature to be 25 ℃, and starting the ultraviolet lamp for sterilization on the sterile operating platform.

2. Craniotomy

(1) Rat anesthesia: anesthesia is performed by injecting 10% chloral hydrate solution into the abdominal cavity.

(2) Rat craniotomy and motor cortex localization: fixing anesthetized rat incisors on a brain positioning instrument maxillary positioner, respectively pushing ear rods on two sides of the positioning instrument into external auditory canals of rats and adjusting heads of the rats to be in the middle. After the scales of the two ear sticks are adjusted to be consistent, the upper nose ring of the tooth fixer is tightly twisted, and the head of the rat is ensured to be fixed. A constant temperature heating pad was placed on the abdomen of the rat and the temperature was kept at about 37 ℃. The scalp was then disinfected with a 75% alcohol cotton swab to prevent infection. A skin incision about 3cm long was cut at the sagittal suture of the head with a pair of surgical scissors, and the subcutaneous tissue was separated. Then 8% hydrogen peroxide solution dipped by a sterilized cotton swab is fully contacted with the surface of the skull, and then fascia tissues and muscles are peeled off by using precision surgical scissors. The periosteum is pushed away gently with tissue forceps, exposing bregma, herringbone and sagittal sutures.

After moving the positioning needle to a sagittal suture, referring to a rat brain positioning chart to move a cortex region, firstly adjusting the positioning needle to bregma, recording the space coordinate of the positioning needle, then adjusting the X axis and the Y axis to be 1mm forward and 1-2mm on the right side of a midline, positioning the positioning needle to the moving cortex, and marking by using a marker pen. And drilling a round hole with the diameter of about 2mm at the marked position by using a skull drill. After the skull was drilled through, the rat dura was carefully teased open and the motor cortex was exposed. Meanwhile, 3-4 small holes are drilled on the skull near the wound for placing skull nails to enhance the fixing effect of the device.

3. Fixation and post-operative treatment of in vivo test cannulas

(1) Cleaning up the periphery of the wound by using a dry disinfection cotton swab, aligning and fixing the 3D printing optical fiber cannula with the bottom coated with glue to the drilled round hole, and simultaneously coating glue on the skull nail and fixing the skull nail in the small hole drilled in advance. Adding 3 spoons of dental cement powder and 1 spoon of carbon powder for polymer black printing into a plastic package bag, uniformly mixing, adding 3mL of II type dental cement self-setting water, and fully mixing. After the dental cement has formed a paste, the dental cement is injected around the skull and cannula. During the injection process, the dental cement is pressed by a wet cotton swab to be uniformly and fully contacted with the skull and the cannula, and a fixed shape is formed. After the dental cement is dried and solidified, the scalp wound is sutured by a suture needle. A sterilized cotton ball is inserted into the opening of the sleeve to prevent other impurities from entering the pipe opening to block and cause infection. And finally, cleaning the experiment table top and cleaning the experiment appliance.

(2) After surgery rats were placed in sterile squirrel cages padded with urine pads of experimental animals and the cages were placed in a warm, constant temperature environment. After the rats were fully awake, they were housed in individual cages in the rat rooms and numbered.

4. Olfactory stimulation and biophoton collection

(1) The device is adaptive to training: each craniotomy rat underwent 3 free explorations of the adaptation device for 15min each time before performing the official olfactory test.

(2) Preparation operation of the ultra-weak biophotonic imaging system: an indoor air conditioner (the temperature is set to be 25 ℃), a dehumidifier (the humidity range is set to be 40% -50%) and a low-temperature cooling circulating pump are started, and the EM-CCD lens is started. And (3) opening the computer, starting the matched operating software (HCimage) of the EM-CCD, setting relevant parameters according to experimental requirements, cooling the EM-CCD system to-90 ℃ and keeping the temperature constant, and ensuring the normal operation of the system. Before placing the rat in the dark box, the device was wiped thoroughly with 75% alcohol and left to dry, avoiding the effects of residual odors. The fiber cannula was then connected to the cannula on the rat skull and the nut was slowly torqued down. And finally, placing the other end of the optical fiber under an EM-CCD lens in a dark box and adjusting the other end of the optical fiber to the center of the lens.

(3) Shooting a location photo: and all indoor light sources are turned off to ensure that the experimental environment is a dark environment. Setting the EM-CCD mode as a Normal mode, setting the exposure time as 1s, clicking a lens Live mode, opening the camera bellows micro-adjusting devices until the best shooting effect can be obtained, and finally shooting a positioning photo.

(4) Collecting biological photons: the Live mode was turned off, the dark box door was closed, and all lights in the room were turned off to ensure the experiment was performed in the dark. The EM-CCD mode was converted to electron multiplication mode with an exposure time of 10s, 90 pictures acquired and a gain level of 1200.

(5) Dark adaptation of rats: and (3) completely turning off the light source of the experimental environment at least 15min before the beginning of the formal experiment, so that the rat adapts to the environment, and the influence on the experimental result caused by physiological change of the rat due to rapid day and night switching is avoided.

(6) Odor stimulation: the dark box is cleaned by 75% alcohol before stimulation, so that the influence of residual smell in the dark box on the experiment is avoided. After the rat is adapted to the dark environment, the rat is placed in a dark box and connected with an optical fiber bundle. And after confirming that the dark box is closed, starting to collect the brain motor cortex biophoton. The test time is 15min, no stimulation is given for 5min before the experiment, and the odor stimulation is given for 6min until the experiment is finished. The next experiment is separated from the last experiment by at least 15 min. The experimental flow is shown in figure 3:

(7) and (3) data storage: and after the experiment is finished, the experimental data is stored to a related path document according to an image format obtained by the EM-CCD. Restoring the parameter setting of the EM-CCD and closing the system and other experimental instruments. And finally, separating the cannula fixed on the head of the rat from the cannula, observing the exposure state of the wound of the rat and the self state of the rat, making an experimental record, and putting the rat back to the rat room for single-cage feeding. Finally, the device was thoroughly cleaned again with 75% alcohol and the laboratory bench was cleaned.

5. Image gray value extraction and data analysis

(1) Extracting an average gray value: because the target area imaged by the optical fiber is small, the image does not need to be processed, white spots caused by cosmic rays are eliminated, and if obvious abnormal signals (caused by cosmic rays) appear in the target area imaged by the optical fiber, the image is subjected to invalid processing. Opening the sequence image by using EM-CCD matched software, importing a positioning picture, selecting a target area by using a software framing tool, and then taking the selected area except the target area as a background area. And finally, extracting the average gray values of the target area and the background area by using a gray value extraction function, and exporting data to a data file (such as Excel) for storage.

Relative gray-scale value (RGV) is target gray-scale value-background gray-scale value

(2) And (3) data analysis: and (2) performing data analysis and statistical analysis on the data file exported in the step (1) by using data analysis software (such as GraghPad Prism).

6. Results of the experiment

Effect of Vanillin on photon radiation from the cerebral motor cortex of female rats

In the experiment, female rats are exposed to the odor environment of vanillin, and the constructed in-vivo ultra-weak biophotonic imaging system is used for recording the characteristic that the odor exposure of the vanillin induces the biophotonic activity change of the cerebral motor cortex of the female rats, and distilled water is used as a control. The experimental sequence chart was imaged every 10 s/frame, with odor exposure occurring at frame 30 of imaging, and ending at frame 90. Imaging time 900 s. Because the delayed luminescence phenomenon exists in the early stage of experimental imaging, the stable interval from the 21 st frame to the 30 th frame is selected in the period before stimulation during statistical analysis. The results of the experiments are shown in FIGS. 4 a-4 f, and FIGS. 5 a-5 f, below.

The experimental results show that the vanillin odor exposure can induce the change of the reduction and then enhancement of the radiation activity of the brain motor cortex biological photon of the female rat to the normal level (figure 5b and figure 5 c). Statistical findings before and after stimulation (fig. 5d, fig. 5 e): the photon radiation activity of the control group and the experimental group is reduced in the 31 th to 45 th frames after stimulation compared with the 21 st to 30 th frames before stimulation, but the control group has significant difference (P <0.05), while the experimental group has very significant difference (P < 0.01); the control group and the experimental group have no difference in 46 th-60 th frames and 61 th-75 th frames after stimulation compared with the pre-stimulation; there was a very significant difference between the 76-90 th frames after stimulation (P <0.01) and before stimulation in the control group, whereas there was no difference in the experimental group.

Statistical analysis of the experimental and control groups at the same time period found (fig. 5 f): the biophotonic radiation activity of the experimental groups was lower than that of the control group both before and after stimulation, and was particularly significantly different (× P < 0.0001). For the ratios shown in Table 1 below

TABLE 1 Change in Motor cortex biophotonic radiation in female rats after stimulation with distilled water and vanillin

Note: the results of relative gray values are expressed as mean ± standard deviation (mean ± s.e.m), with distilled water n being 10 and vanillin n being 8. Statistical methods were Unpaired T-tests (un-paired T-test), P <0.05, P <0.01, P <0.001, P < 0.0001.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims. Thus, the present discovery is a general technique that requires protection.

Claims (7)

1. An in vivo animal brain biological photon imaging detection system comprises an ultra-weak biological photon imaging system UBIS, a movable box, an optical fiber bundle and an optical fiber bundle fixing sleeve device; the method is characterized in that: the optical fiber bundle fixing sleeve device is fixed on the skull of an animal to be tested, one end of the optical fiber bundle is fixed below a photon imaging device in a UBIS dark box of the ultra-weak biological photon imaging system, and the other end of the optical fiber bundle is connected with the optical fiber fixing sleeve device placed on the skull of the animal to be tested in the movable box.

2. The in vivo animal brain biophotonic imaging detection system of claim 1, wherein: the stimulation device is arranged in the movable box and used for stimulating the animal to be tested.

3. The in vivo animal brain biophotonic imaging detection system of claim 2, wherein: the stimulation device is a Bluetooth intelligent device and can perform stimulation including light, sound, smell, electricity, heat and/or medicines.

4. The in vivo animal brain biophotonic imaging detection system of claim 1, wherein: the optical fiber bundle fixing sleeve device is composed of an insertion pipe, a sleeve and a nut, wherein one end of the insertion pipe is provided with a platform, one end of the sleeve is provided with an external thread, the nut is provided with an internal thread, one end of the optical fiber bundle is connected with the insertion pipe, the insertion pipe penetrates through the nut and the platform to be clamped in the nut, the nut and the sleeve are tightly fixed through the thread, the other end of the sleeve is fixed on the skull of an animal to be detected, and the bottom of the sleeve is an exposed cerebral cortex area of the animal to be detected.

5. The in vivo animal brain biophotonic imaging detection system of claim 1, wherein: the optical fiber bundle is formed by bonding more than 9 optical fibers into a bundle by using an adhesive, and two ends of the optical fiber bundle are respectively embedded with a ceramic ferrule; all optical fibers are sleeved in a black protective sleeve, and the two ends of the protective sleeve are bonded with the metal tail handle of the ceramic ferrule and are wrapped and shielded from light.

6. The in vivo animal brain biophotonic imaging detection system of claim 5, wherein: the optical fiber is a multimode or single-mode optical fiber.

7. The detection method using the in-vivo animal brain biophoton imaging detection system as claimed in claims 1-6 is characterized by comprising the following steps:

(1) the experimental preparation operation of the ultra-weak biophoton imaging system comprises the following steps: opening an indoor air conditioner, a dehumidifier and a low-temperature cooling circulating pump, setting the temperature to be 25 ℃ and the humidity to be 40-50%, and starting an EM-CCD lens; opening a computer, starting the matched operating software of the EM-CCD, setting relevant parameters according to experiment requirements, cooling the EM-CCD system to-90 ℃ and keeping the temperature constant, and ensuring the normal operation of the system; finally, the optical fiber bundle is integrally arranged in an EM-CCD dark box, one end of the optical fiber bundle is arranged below an EM-CCD lens and is adjusted to the center of the lens and is fixed, and the other end of the optical fiber bundle is connected with an optical fiber fixing sleeve device arranged on the skull of the animal to be detected in the movable box;

(2) shooting a positioning photo: turning off all indoor light sources to ensure that the experimental environment is a dark environment; setting the EM-CCD mode as a Normal mode, setting the exposure time as 1s, clicking a lens Live mode, opening a camera bellows door to finely adjust an EM-CCD camera lens until the best shooting effect can be obtained, and finally shooting a positioning photo;

(3) collecting biological photons: closing the Live mode, tightly closing a dark box door, and turning off all indoor light sources to ensure that the experiment is carried out under the dark condition; converting the EM-CCD mode into an electron multiplication mode, setting the gain level to be 1200, and setting exposure time and the number of collected pictures corresponding to the experiment requirements;

(4) and (3) data storage: after the experiment is finished, the experimental data is stored to a relevant path document according to a certain data format; restoring the parameter setting of the EM-CCD and closing the system and other experimental instruments;

(5) extracting an average gray value: performing bright spot removal processing on the sequence diagram by using a well-written program on a software platform, and avoiding the influence of cosmic rays on the extraction of the gray value of the image; opening the sequence image by using EM-CCD matched software, importing a positioning picture, selecting a target area by using a software framing tool, and then taking the selected area except the target area as a background area; finally, extracting the average gray values of the target area and the background area by using a gray value extraction function, and exporting data to a data file for storage;

(6) and (3) data analysis: and (5) performing data analysis and statistical analysis on the data file exported in the step (5) by using data analysis software.

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