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1. Application of in vivo fluorescence microscopy imaging to observe rat brain neurons
Rats were anesthetized with 7% chloral hydrate (0.6 mL/100 g) by intraperitoneal injection, shaved and fixed on a stereotaxic instrument. Exposing the pre-existing skull hole, opening the volumetric fluorescence microscopy imaging system, inserting the soft fiber ProFlex with a diameter of 650 μm in the volumetric fluorescence microscope vertically into the rat brain, passing through the meninges, and vertically inserting the needle into the PtA region. The injection site was excited by a 488 nm laser, and the acquired signal band was 500-650 nm, and the acquisition frequency was 12 frames/s recorded in the body fluorescence image (Fig. 1). 
2 results
2.1 In vivo fluorescence microscopy imaging of rat brain
Fluorescence images of the target location of the fiber optic probe can be acquired in real time by the in-vivo fluorescence imaging system. In the microinjection site of the rat brain, the fiber probe was fine-tuned, and two fields of view were selected for observation and the dynamic changes of the fluorescence signals of GFP-labeled neurons were recorded for 10 min. Using the IC-Viewer software, the recorded data can be exported in an image or image format. Figures 2A and B show six consecutive screenshots of 10 s from the two data collected from the same sample. In the figure, multiple GFP-labeled intracerebral neurons at the corresponding time points can be observed. Morphology, GFP of neuronal cell bodies
The fluorescent signal is more obvious. There was no significant change in the morphology of GFP-labeled neurons during the recorded time period. 
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The probe-type in vivo fluorescence microscopy imaging technology combines the two techniques of microfiber probe and laser scanning [8] to realize the function of real-time tracking and recording of dynamic changes in signals in animals. Moreover, because the probe is only 650 μm in diameter and the material is soft, it can meet the requirements of minimally invasive surgery. The probe consists of nearly 30,000 soft fibers (Figure 4) with an observation range of 600 μm × 500 μm, an axial resolution of 15 μm, a lateral resolution of 5 μm, and high sensitivity, so it can be used in the nerve In vivo tracking of neuronal signals in animal brains in the scientific field [9]. In vivo fluorescence microscopy
The technology is excited by a 488 nm laser, and the acquisition signal band is 500~650 nm, and the acquisition frequency is 12 frames/s. The signal code collected by the instrument is 13 bits. The software can export images in various formats such as png, bmp, and jpeg, as well as images in mpeg and mhd (raw format) formats. The morphology of GFP-labeled neurons in rat brain was observed in real time by in vivo fluorescence microscopy imaging. The continuous screenshots within 10 s show that although the physiological factors such as respiratory and vascular beating of living animals have a slight effect on the stability of the image, the intercepted images of the target neurons in a short time are relatively stable, and the neuronal cell fluorescence signals are relatively stable. It is clear and consistent with the literature [5]. This also suggests that no significant phototoxicity was produced during the recording process. The brain tissue of the same rat was frozen and sectioned, and observed under a fluorescence microscope. The GFP-labeled neurons were observed, and the objective fluorescence imaging technique was further confirmed to be objective and effective.
The results show that the probe-based fluorescence microscope can stably record the morphology of neurons in a specific field of view over a period of time, so it can be combined with stereotactic techniques for observation of neuronal development and neuronal migration. In-vivo fluorescence imaging technology combines image analysis software with its real-time image acquisition software. In addition to real-time observation of morphological changes in vivo neurons, it can also perform rapid response phenomena such as changes in calcium flux in the body. Dynamic data collection [5] can also analyze tissue structures other than neurons, such as vascular indicators, and even synchronous acquisition with other physiological information in the body. The corresponding incision fixing fittings can be used to complete the data recording in the animal awake state.
In addition, since the excitation wavelength of the fiber-optic probe is 488 nm, it can also be used for precise localization to activate ChR2 photoprotein in optogenetics.
In summary, fiber-optic in vivo fluorescence microscopy imaging provides more room for expansion in our neuroscience experiments.
This article is an excerpt from Shi Ying, Institute of Brain Science, Fudan University, and State Key Laboratory of Medical Neurobiology, Chen Luzhen, Jiang Min et al., Acta Physiologica Sinica, December 25, 2012, 64(6): 695–699
Shi Ying, Chen Luzhen, Jiang Min, et al., Acta Physiologica Sinica, December 25, 2012, 64(6): 695–699, Department of Brain Science, Fudan University, Shanghai, China An experimental method for the continuous observation of in vivo microscopic fluorescence of rat brain neurons was established. The study began to microinject the recombinant viral vector containing green fluorescent protein (Ubi-GFP) into the cerebral cortex of Sprague Dawley (SD) rats. After 7 days, the fiber probe was implanted into the target position of the animal brain by minimally invasive surgery. Specific fluorescence signals of GFP-labeled neurons in the microinjection zone were observed by fluorescence microscopy imaging. Subsequently, the frozen brain sections of the rat brain tissue were examined by fluorescence microscopy. The fluorescence signals observed in the bulk fluorescence microscopic imaging experiment were verified. The method not only ensures the physiological state of the animal in the body experiment, but also satisfies the requirements of the fluorescence microscopic level imaging of the nerve tissue in the body, and can be used for the in vivo tracking recording of the neuronal fluorescence signal in the animal brain, which is an in vivo neuroscience experiment. Provides effective technical support.