Felh1300

Figure 1a shows the NIR-II fluorescence stereo system, featuring epi-illumination geometry and fiber-based configurations with a fluorescence signal excited by a 793 nm continuous-wave fiber laser (CNI laser, FC-W-793). Then, light emitted from fiber (MHP550L02, Thorlabs, Newton, NJ, USA) was transmitted via a ground glass diffuser (Thorlabs, DG10-600-MD) providing uniform illumination in the irradiation area (50–70 mW/cm²). For whole-body imaging of the mouse, long-pass (LP) filters (FELH1100, FELH1300, and FELH1400, Thorlabs) were employed, while in vivo images were acquired using a cooled InGaAs camera (NIRvana 640, Princeton Instruments; 640 × 512 pixels, response 900–1700 nm) with a short-wave infrared C-mount zoom lens (LM35HC-SW, Kowa, Tokyo, Japan). The working temperature of the InGaAs camera was −80 °C, the gain was set in high mode, and the analog-to-digital conversion rate was set at 2 or 10 MHz. A camera and a one-dimension moving stage were used for NIR-II stereo imaging (Figure 1b). The images were acquired with LightField software (Princeton Instruments, Trenton, NJ, USA) and analyzed with MATLAB (MathWorks, 2020).

The layout of the proposed system followed a generalized widefield configuration adapted to provide stereovision performance by using two tilted cameras (Figure 1a). A continuous‐wave 855 nm laser diode was positioned between the two cameras to provide uniform epi‐illumination. Fluorescence responses were collected in dual‐view mode with the two cameras. Specifically, the two detection modules were fixed at ±20° symmetrically with respect to the main axis (vertical direction), each containing a 1300 nm long‐pass filter (FELH1300, Thorlabs, USA), a camera lens (LM50HCSW, 50 mm effective focal length, Kowa, Japan), and an InGaAs‐based SWIR camera (WiDy SenS 640V‐ST, NiT, France) mounted on an adjustable rotation stage (RP01/M, Thorlabs, USA). The magnification ratio of the detection system was set to ≈0.37. The pixel pitch on the camera sensor was 15 µm, corresponding to 40.5 µm in the object plane. For dual‐view recordings, the cameras were operated in a linear mode and synchronized with an external trigger signal. A non‐uniformity correction file was collected for each camera under specific exposure time and temperature, and subsequently applied to provide optimal image quality.