研究目的
Investigating the use of dual-modal fluorescence and photoacoustic microscopy for noninvasive and functional in vivo imaging of inflammation induced by GFP-expressing bacteria in mice ear to monitor tissue immunovascular responses and track biochemical changes.
研究成果
The dual-modal imaging approach effectively monitors inflammation by combining the strengths of fluorescence microscopy for bacterial tracking and photoacoustic microscopy for vascular changes. It provides high resolution and sensitivity, enhancing the understanding of inflammatory pathogenesis. Future work could focus on improving imaging depth and applying the technique to other disease models.
研究不足
The study is limited to small animal models (mice ears), and the imaging depth and resolution may not be sufficient for larger tissues or clinical applications. Fluorescence imaging cannot identify normal tissue structures without infection, and PAM may not effectively capture low-absorption targets like bacteria. The method requires specialized equipment and may not be easily scalable.
1：Experimental Design and Method Selection:
The study employed a dual-modal imaging approach combining homemade optical-resolution photoacoustic microscopy (PAM) and commercial fluorescence microscopy to image inflammation in mice ears. The rationale was to leverage complementary information from both techniques for high-sensitivity detection and monitoring of inflammation.
2：Sample Selection and Data Sources:
GFP-transfected E. coli bacteria were cultured and used to induce inflammation in six eight-week-old nude mice. The mice ears were imaged before and after bacteria injection.
3：List of Experimental Equipment and Materials:
Equipment included a homemade PAM system with a pulsed laser (532 nm wavelength, 1 ns pulse width, up to 5 KHz repetition rate), optical components (neutral density filter, convex lenses, pinhole, optical fiber, objective lenses, rectangular prism), a two-dimensional motor stage, an ultrasonic transducer (VF 412, Valpey Fisher), an amplifier (5073R, OLYMPUS), and a commercial fluorescence microscopy system (eclipse Ni-U, Nikon Inc.) with a mercury lamp, achromatic condenser, and CCD camera. Materials included LB medium, PBS (Gibco, Thermo Fisher Scientific), agar gel phantom, ultrasound gel, syringes, needles, and anesthesia machine (Isoflurane, R580, RWD Life Science).
4：Experimental Procedures and Operational Workflow:
GFP-transfected E. coli were cultured, washed, and suspended in PBS. Mice were anesthetized, and their ears were imaged before injection. Bacteria were injected into the ear, and imaging was performed at 6 hours post-injection and over 5 days. The PAM system involved laser illumination, signal detection with the transducer, amplification, and image reconstruction. The fluorescence system used excitation at 488 nm and emission filters at 525-550 nm with an integration time of 100 ms.
5：Data Analysis Methods:
Fluorescence intensity was quantified using a microplate reader and analyzed to correlate with bacterial concentration. Photoacoustic and fluorescence images were compared to assess immunovascular responses and bacterial distribution.
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