With the rapid development of life sciences and biotechnology, the demand for observing the structure and function of living organisms has been increasing. Common imaging methods such as nuclear imaging, magnetic resonance imaging, and computed tomography have been unable to accurately and continuously monitor living organisms due to issues such as radiation and expensive equipment. Although slice detection is highly accurate, it cannot directly observe living organisms. Fluorescence imaging works by marking specific molecules within cells or body fluids with fluorescent probes, making the fluorescence signal much stronger than the organism's own luminescence, thereby enabling the observation and measurement of target tissues. However, current fluorescence in vivo imaging instruments mainly focus on single-scale detection. If multi-scale in vivo detection that combines macroscopic and microscopic scales can be achieved, it will have significant implications for the rapid detection of human diseases.Near-infrared II (NIR-II, 900-1880 nm) based in vivo fluorescence imaging technology is currently a cutting-edge in vivo biological imaging technique, which offers a deeper tissue penetration depth. This method first genetically induces live laboratory rat to become fluorescent live laboratory rat through the iRFP713 fluorescent protein. To locate the diseased organs, the laboratory rat can be placed under the macroscopic imaging function module for observation. After analyzing and identifying the target area of interest, the live animal is pushed into the microscopic imaging function module for microscopic observation. Through scanning and image stitching, a full-field microscopic image is obtained. Additionally, by traversing and photographing the microscopic images and then using image stitching, a full-field microscopic image can be synthesized, thereby achieving non-invasive and rapid detection of live animals.The experiments of macroscopical imaging and microscopical imaging in living laboratory rat were carried out by using multi-scale NIR-II fluorescence imaging method. Macroscopic: Figure 3(a) shows live laboratory rat without fluorescent liver. At this time, the camera exposure time is 250 ms, and the laser power is 150 mW. Figure 3(b) shows live laboratory rat with fluorescent liver. At this time, the camera exposure time is 90 ms and the laser power is 150 mW. It can be seen that the liver in vivo showed a relatively complete state under the observation of this system, especially the concave flap structure in the middle of the liver. Microscopic: Scanning and photographing in Z-shape step by step with the lens, 180 images were obtained. As shown in Figure 3(d), 180 images were spliced and the microscopic global image of the liver was shown in Figure 3(e). The approximate location of the liver in the laboratory rat is shown in Figure 3(f). Objects of different scales can be clearly identified, and the function of the system meets the needs of multi-scale imaging.To address the scale limitation of existing NIR-II fluorescence imaging instruments to a single scale, this paper proposes a multi-scale NIR-II fluorescence in vivo imaging method and develops an experimental verification device to verify the feasibility of the method. The method is as follows: We take the liver of an experimental laboratory rat as the observation object. First, we conduct macroscopic observation of the live laboratory rat to obtain a large field-of-view image and identify the organ with lesions. To obtain further information about the lesion, we then perform microscopic in vivo imaging of the organ to obtain microscopic images of the lesion organ in the live experimental laboratory rat. By stitching the images, we can obtain an image data set with higher information density than macroscopic in vivo imaging, facilitating the subsequent collection of specific lesion information. The multi-scale imaging system we built has a large imaging field-of-view and high imaging resolution. The macroscopic imaging field-of-view can reach 192 mm × 154 mm, with a resolution of 300 μm; the microscopic imaging range can reach 3.2 mm × 2.56 mm (5× objective lens), 0.8 mm × 0.64 mm (20× objective lens), with a resolution of 5 μm (5× objective lens)/1.25 μm (20× objective lens). The final experimental image data show that under the macroscopic imaging function, the fluorescent liver of the live laboratory rat can be clearly imaged, and the macroscopic contour of the liver can be distinguished; under the microscopic imaging function, the details inside the liver can be observed, and microscopic whole-field imaging can be achieved by using image stitching technology.