, where [Fluo] i and [Exc] i represent the fluorescence and excitation image for source position i, respectively, and s is a smoothing factor that prevents division by very small excitation values). This resulting image does not provide true quantitative 2D but should correlate spatially with the depth projection of the results obtained in the reconstruction.
2.
Quantify 3D fluorophore concentration. If the system has been properly calibrated for imaging the fluorescent probe used, the 3D images should be quantitative (see Note 22 ). This is done by using mathematical algorithms to solve the “inverse problem.” Some of them are described in [1, 3, 4]. Commercial systems include their own proprietary software for 3D reconstruction of the data (see Note 13 ).
3.
Unmix spectra. As mentioned previously (see Notes 11 and 14 ), whenever the system allows the acquisition of data from several excitation/emission pairs, spectral unmixing helps in reducing the contribution of autofluorescence (see Note 23 ) and allows imaging of more than one fluorophore simultaneously, even if there is overlap between the emission curves. It is important to understand that spectral unmixing will not help in elucidating which is the probe that has been activated or bound specifically to the unspecific activation or unbound probe, since both present the same emission spectrum.
3.5 Validation by Histology
It is recommended to validate the results of FMT by performing postmortem histological analysis.
1.
Euthanize the animal following the procedures accepted by the ethics committee.
2.
Surgically remove the area of interest (i.e., the aortas), embed it in Tissue-Tek (OCT compound), and freeze it at −80 °C.
3.
Cut cryo-sections using a cryotome and mount them in glass slides, covered by a small drop of mounting medium and a coverslip.
4.
Acquire fluorescent images of the frozen sections with an epifluorescence microscope connected to a digital camera.
4 Notes
1.
Among the existent mouse models for atherosclerosis, low-density lipoprotein receptor-deficient mice (LDLR−/− mice) and apolipoprotein E-deficient mice (apoE−/− mice) are the most widely used. LDLR−/− mice show delayed clearance of VLDL and LDL from plasma, developing atherosclerosis on high-fat diet. On the other hand, homozygous deficiency in apoE contains the entire spectrum of lesions observed during atherogenesis and was the first mouse model described to develop lesions similar to those of human. Under normal dietary conditions, apoE−/− mice develop extensive atherosclerotic lesions, and the process can be accelerated on a high-fat diet (see Note 2 ). A chronological analysis of atherosclerotic lesions in apoE−/− mice revealed that the sequential events involved in lesion formation in this model are strikingly similar to those in larger animal models and in humans [16].
2.
High-cholesterol and high-fat diets are used to accelerate the formation of atherosclerotic plaques in mouse models of atherosclerosis. The atherosclerosis-promoting diet most frequently used in mice contains 0.15 % cholesterol and 21 % fat derived from milk fat. After 10–14 weeks under this diet, apoE-deficient mice develop complex fibrous plaques in the aortic sinus [16, 19].
3.
Providing chlorophyll-free chow to the animals is recommended at least 10 days prior imaging, in order to reduce autofluorescence [20].
4.
Human exposure to anesthetic gases has been associated with health risks. Therefore, safety considerations have to be taken into account when using inhaled anesthesia.
5.
Applying an ocular lubricant cream in the eyes of the animals during anesthesia is recommended in order to prevent corneal desiccation. Avoid touching the eye with the tip of the ointment dispenser as it may scratch the cornea. Using a small piece of tissue paper to apply the cream is recommended.
6.
Hair strongly interferes with light, and therefore hair removal from the skin is highly recommended for FMT experiments when using haired animals. In black haired mice, such as the C57BL/6 strain, the first steps of hair growth are accompanied by strongly dark pigmentation of the skin [21]. This pigmentation will completely ruin the FMT experiment due to light absorption. Therefore, whenever performing a longitudinal study with black haired mice, the time between hair removal and the initiation of skin pigmentation should be considered as the optimal imaging period. Whenever possible, it is recommended to use albino mice (such as albino C57BL/6 mice, which carry a mutation in the tyrosinase gene).
7.
Hair removal creams are very irritant for the skin of the mice. They should not be applied for longer than 1 min. We recommend diluting it in lukewarm water (1:2) prior to using it and wash it away right after shaving, keeping always the animal warmed on a heating pad.
8.
9.
Fluorescent imaging agents are usually administered intravenously. Exceptional cases may require administration via intraperitoneal.
10.
Placing the animal in an adequate holder during the imaging procedure is the key to obtaining good quality images. The animal holder of each imaging system might be slightly different; ensure that good coupling between the light source and the exposed tissue takes place in order to avoid the contribution of stray light (i.e., light that has not traveled through the animal) to the detected signal.
11.
Depending on the FMT system employed, several laser lines will be available. Choose the laser line and emission filter most appropriate for the probe under study. Whenever imaging more than one probe, if the setup allows it, take as many combinations of excitation/emission pairs as possible, always making sure that the emission filter used covers a range of wavelengths longer (i.e., lower energies) than the excitation source, always bearing in mind the total duration of the experimental session. Before ordering a fluorescent probe, make sure the FMT setup you have access to has the optimum excitation/emission pair for this particular probe: not all FMT systems have the same laser lines and filters, and most commercially available ones do not provide imaging below 600 nm.
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