We have shown that autofluorescence and SHG emissions can be excited and imaged by multiphoton microscopy in acute brain slices of transgenic AD mouse models. More specifically, we confirmed that senile plaques exhibit autofluorescence with a distinct emission spectrum and also weakly generate SHG. This autofluorescence was seen in all four of the transgenic mouse models examined; therefore suggesting this type of intrinsic emission is a general property. This ability to identify senile plaques by their autofluorescence enables an array of possible functional studies using other intrinsic emissions, which we demonstrated by imaging SHG from dendritic microtubule arrays near senile plaques.
Previous studies that use multiphoton microscopy in AD mouse models focus on imaging dye-stained senile plaques. In a pioneering study [30
], Christie et al. developed a thinned skull preparation in mouse that allows for imaging of senile plaques over several months. This success was followed by other in vivo
time-lapse studies, which use other complementary techniques such as neurite tracing [26
], calcium imaging [31
], astrocyte stains [32
], or direct application of drugs and antibodies [33
] to obtain additional insights on the disease. Here, we showed that weaker intrinsic emissions can also be imaged effectively in AD mouse tissues. This opens the door for new approaches for functional imaging of other intrinsic emissions, such as NAD(P)H metabolic imaging, to be used to study the pathological mechanisms of AD.
Although not demonstrated here, it may be possible to use autofluorescence as a diagnostic tool for in vivo
studies in transgenic mouse models. Our study shows that autofluorescence from senile plaques is weak but clearly detectable and has a characteristic multiphoton-excited emission spectrum. One possible diagnostic method deep within the brain is to use gradient-index (GRIN) lenses [34
] or optical fiber bundles [36
] to excite and collect autofluorescence from a small volume within the brain. Imaging may not be required, since spectral information will be the key for distinguishing AD versus normal tissues.
In our imaging-only results, senile plaques and lipofuscin have broad emission spectra so we distinguish them by morphology. In terms of size, senile plaques have diameters ~10 times larger than lipofuscin. This size difference can be clearly resolved in multiphoton microscopy images, which have submicron spatial resolution within scattering tissues. In some cases, lipofuscin can “clump” together to form larger structures. However, in those cases, multiphoton microscopy images can still clearly resolve individual, smaller-diameter lipofuscins (see Fig. 4(a) in [4
]). On the other hand, it is unlikely to find small, nascent plaques. Plaques grow quickly in vivo
within 1–3 days to their full size [30
]. Therefore, finding nascent plaques is a rare occurrence even in animal models with significant plaque load [37
]. Although morphology may be a good correlate, identification of senile plaques versus other autofluorescent materials would be more accurate if spectral information were obtained.
We have characterized the emission spectrum of senile plaque autofluorescence. Previous studies [9
] have used excitation and detection at a variety of wavelengths, so knowledge of the emission spectrum will be useful for more sensitive detection. As a quick test to find the molecular origin of this autofluorescence, we tested solutions of A-beta fibrils, in which fibrils were verified with transmission electron microscopy, and saw no one-photon-excited fluorescence in a standard fluorimeter. This initial observation suggests that senile plaque autofluorescence is unlikely to originate from A-beta, which is the principal component of senile plaques [38
]. Many other materials are known to exist within senile plaque [38
] and can contribute to the intrinsic emission.
Autofluorescence from NFTs has been described before in post-mortem human tissues [4
], but was not observed in the APPSwe/PS1/Tau models in this study. A previous report [16
] has shown that these mice are capable of generating Thioflavin-S-positive tau-related lesions. The absence of autofluorescence from NFTs in our work could be due to two reasons: One, the previous report [16
] observed Thioflavin-S-positive lesions in 12-month old homozygous mice, which may be at a more advanced stage of AD than our ~20-month old heterozygous mice. This argument is supported by the observation that Thioflavin-S did not stain any structures that resemble tau-related lesions in our acute slices. Two, it is possible that the only types of human tau pathology that exhibit autofluorescence have no analogs in the APPSwe/PS1/Tau transgenic mouse model. To distinguish between these possibilities, more research would be required on characterizing autofluorescence and immunoreactivity from various kinds of tau-related lesions in human and transgenic mouse model tissues.
Intrinsic emissions such as autofluorescence and SHG are useful indicators for detecting the presence of pathological lesions. Changes in the intensity and spectrum of intrinsic emissions have previously been studied as possible diagnosis methods for tumors [39
] or skin pathology [40
], and can potentially also be useful for neurodegenerative diseases. Here we have demonstrated that intrinsic emissions, particularly that of senile plaques, can be detected from relatively thick, native tissues. Furthermore, the senile plaque emission spectrum is distinct from the background to possibly further enhance detection sensitivity. We anticipate that this work will be useful for interpreting future studies that aim to use endogenous optical signals as a diagnostic tool or as functional fluorescent indicators.