Fesh0900

A home-built system was established to perform ASF wide-field microscopic imaging of cerebral vessels in mice. 915 nm CW laser beam as the excitation light source passing a 900 nm long-pass filter (FELH0900, Thorlabs) was incident on a 900 nm short-pass dichroic mirror (#69-222, Edmund Optics) and then reflected, irradiating onto the mouse brain through an infrared antireflection water immersion objective (XLPLN25XWMP2, 25×, NA = 1.05, Olympus). The excited ASF was collected by the same objective, and then passed through the same 900 nm short-pass dichroic mirror and a combination of a 900 nm short-pass filter (FESH0900, Thorlabs) and an 800 nm long-pass filter (FELH0800, Thorlabs). Finally, ASF signals were focused on a wide spectral responsive Si-based camera (GA1280, 1280 pixels × 1024 pixels, TEKWIN SYSTEM, China) through the built-in tube lens in the trinocular to visualize the cerebral vessels of the mouse. The system was equipped with an electric control module, which could control the objective (together with the whole microscope unit) to move along the Z-axis direction and collect the signals at different depths of the mouse brain for depth tomography. It could also control the loading platform to move in the X-Y direction and change the imaging field of view.

The 915 nm CW laser beam was coupled to a collimator through the optical fiber, and then expanded by a lens with a ground glass sheet which could eliminate laser speckles to provide a large illumination area. There were two sets of imaging systems placed parallel to each other, and one was the ASF imaging channel. Fluorescence signals were captured by a wide spectral responsive Si-based camera (GA1280, 1280 pixels × 1024 pixels, TEKWIN SYSTEM, China) after passing through the prime lens (focal length: 35 mm) with an antireflection coating at 800–1700 nm and a combination of a 900 nm short-pass filter (FESH0900, Thorlabs) and an 800 nm long-pass filter (FELH0800, Thorlabs) filtering away the excitation light. The other was the SF imaging channel, which consisted of the same prime lens, a 1100 nm long-pass filter (FELH1100, Thorlabs), and an InGaAs camera (640 pixels × 512 pixels, TEKWIN SYSTEM, China). The two sets were next to each other, and the rat was placed in their common field of view.

This system is divided into two parts. One part is used for fluorescence imaging and the other part is used for photothermal treatment. For imaging, 915 nm CW laser beam for ASF excitation and 793 nm CW laser beam for SF excitation were separately coupled to two collimators through two optical fibers. The 793 nm laser beam was reflected by an 805 nm long-pass dichroic mirror (DMLP805R, Thorlabs) while the 915 nm laser beam was transmitted through the dichroic mirror. Next, either beam could be expanded by a lens with a ground glass sheet, which was used to eliminate laser speckles and provide a large illumination area. Fluorescence signals from tumors of mice were captured by a wide spectral responsive Si-based camera (GA1280, 1280 pixels × 1024 pixels, TEKWIN SYSTEM, China) after passing through the prime lens (focal length: 35 mm) with an antireflection coating at 800–1700 nm and a combination of a 900 nm short-pass filter (FESH0900, Thorlabs) and an 850 nm long-pass filter (FEL0850, Thorlabs) filtering away the excitation light. For photothermal treatment, 1550 nm CW laser beam was coupled to a collimator (F810FC-1550, Thorlabs) through the optical fiber, and then the collimated laser beam irradiated on the subcutaneous breast tumor of the mouse. A thermal imager (TiS20, FLUKE) was used to record the precise temperature in photothermal treatment.