A custom-built setup consisting of a fiber (200 µm core) coupled 793 nm laser (20 W, Lumics LU0793D140-D10DH) was used. The laser is launched in an excitation cube by the fiber and reflected through a mirror (Thorlabs BBE1-E03). The reflected light is passed through a positive achromat (Thorlabs AC254-050-B) to an engineered diffuser (Thorlabs ED1-S20-MD) to provide a uniform illumination over the working area. The average excitation flux at the animal was close to 15 mWcm−2 (± 3%). The working area was covered with a blackout fabric (Thorlabs BK5). Fluorescence signals were directed to a 4-inch square first-surface silver mirror (Edmund Optics, 84,448) and then to a filter set (Thorlabs, 2 × FELH01000). At the end, the filtered signals were collected using an Allied Vision Goldeye G-032 Cool TEC2 camera with sensor set point at − 30 °C and equipped with a C-mount lens (Navitar, SWIR-35). The assembly was partially enclosed to avoid exposure to unwanted light. The images were acquired using platform independent Vimba SDK (Allied Vision) with a uniform acquisition time of 10 ms. During imaging mice were placed in supine position over pre-defined area under the influence of isoflurane. Inflammation in the tail was induced by intramuscular injection of histamine (0.02 mg/ml). MATLAB programming environment was used for processing and analysis of the acquired images.
Ac254 050 b
Manufactured by Thorlabs
The AC254-050-B is a 50 mm focal length plano-convex lens made of BK7 glass. It has a 25.4 mm diameter and an anti-reflection coating optimized for the visible spectrum. This lens is designed to converge or diverge collimated light.
Automatically generated - may contain errors
3 protocols using ac254 050 b
1
Fluorescence Imaging of Histamine-Induced Inflammation
2
Whole Mouse Imaging with Multiwavelength Excitation
3
Whole Mouse Imaging with Multiwavelength Excitation
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For whole mouse imaging, a custom-built setup was used. Lumics laser units: LU1064DLD350-S70AN03 (35 W) “1064 nm”; LU0980D350-D30AN (35W) “980 nm”, and LU0785DLU250-S70AN03 (25 W) “785 nm” were used for excitation. Laser modules are specced to ± 10 nm. Laser outputs were coupled in a 4 × 1 fan-out fiber-optic bundle (Thorlabs BF46LS01) of 600 μm core diameter for each optical path. The output from the fiber was fixed in an excitation cube (Thorlabs KCB1EC/M), reflected off of a mirror (Thorlabs BBE1-E03), and passed through a positive achromat (Thorlabs AC254–050-B), SP filter (if necessary) and a ground glass diffuser (Thorlabs DG10–120-MD) or an engineered diffuser (Thorlabs ED1-S20-MD) to provide uniform illumination over the working area. In a typical experiment, the excitation flux at the object was adjusted to be close to 100 mWcm−2 with an error of ± 3% (power density used is defined separately in each experiment). The working area was covered by a heating mat coated with blackout fabric (Thorlabs BK5). Emitted light was directed onto an Allied Vision Goldeye G-032 Cool TEC2 camera with a sensor temperature set point of −20 °C.
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