The availability of transgenic mice expressing FRET biosensors prompted us to visualize protein kinase activity in living animals by intravital imaging. For this purpose, we used two-photon excitation microscopy (TPEM), which allows deeper tissue penetration of the light and less phototoxicity than the conventional single-photon confocal microscopy (Molitoris & Sandoval 2005). Intravital imaging with TPEM has been applied to a wide range of tissues and organs in mice, including the brain (Noguchi et al. 2011; Grienberger & Konnerth 2012), auricular skin, blood vessels (Egawa et al. 2011; Kamioka et al. 2012), lymph nodes (Okada et al. 2005; Cahalan & Parker 2008; Kitano et al. 2011), cecum (Toiyama et al. 2010; Tanaka et al. 2012), intestines (Mcdole et al. 2012), bone (Ishii et al. 2009), lung (Looney et al. 2011), liver, pancreas, kidney (Ashworth et al. 2007; Camirand et al. 2011), heart (Li et al. 2012), muscles (Cao et al. 2012), and cancers (Kedrin et al. 2007; Le Devedec et al. 2011). Here, we will briefly review general procedures and tips for intravital FRET imaging with TPEM, although the techniques required for TPEM are heavily dependent on the particular tissues or organs targeted. First, the mice are anesthetized with volatile anesthetic isoflurane. Next, the target tissue is exposed by hair removal and/or surgical operation. The mice are placed on a microscope stage maintained at 30°C using a heating pad. To minimize motion artifacts ascribed to heart-beating and breathing of mice, the object to be imaged should be moved away from sources of motion, such as the heart (Fig. 4b). Laser ablation or intravenous administration of drugs is easily applied for in vivo perturbation under the condition of intravital imaging (Fig. 4c,d). For FRET imaging with TPEM, we use a wavelength of 840 nm in two-photon excitation for CFP. Importantly, although we do not know the reason at this moment, a slow scan speed (10~100 us/pixel) and a low voltage of the photomultiplier tube decrease the noise of CFP and FRET signals, resulting in an increase in the signal-to-noise ratio of the FRET/CFP value, which is a quantitative measure of FRET images (see below). It should be noted, however, that slow scan speed makes the image sensitive to motion effect of heart beat or breathing, and also causes generation of heat and photo-bleaching. Therefore, slowing the scan speed is a trade-off of these disadvantages. In general, long intervals of time-lapse imaging avoids heat generation. Intravital imaging appears to be more resistant to heat generation than ex vivo and in vitro imaging, because of the existence of blood circulation. The problem of photo-bleaching should be examined carefully by trial-and-error to see whether photo-bleaching takes place in that microscopic setting. A simple way is the reduction of laser power. This could be achievable, because the slow scan speed makes it possible to detect more photons than fast scan speed.
For successful intravital imaging with TPEM, it is important to maximize the collection efficiency of scattering fluorescence generated at the focal point of two-photon excitation. In order to take full advantage of the depth penetration of TPEM, the use of external detectors, which are pinhole-less detectors located close to the specimen to reduce the optical elements and light path length, and an objective lens with the highest numerical aperture and field number, are best suited. This leads to the question of whether an upright microscope or an inverted microscope is preferable for intravital imaging with TPEM. In fact, each microscope has its advantages and disadvantages. With the upright microscope, micropipettes are accessible to the observed area of the specimen. Currently, objective lenses customized for TPEM, that is, lenses with high numerical apertures, high field numbers and optical transparency for infra-red light, are usually designed for the upright microscope. However, it is difficult to stabilize the animal under the upright microscope, and therefore samples are susceptible to motion artifacts. On the other hand, with the inverted microscope, the animals can be held down on the cover glass, minimizing motion artifacts. These advantages and disadvantages for upright and inverted microscopes should be considered carefully.