Optical visualization of Alzheimer’s pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation.

2.1.

Multiphoton and second harmonic microscope

Imaging was performed on a custom-built multiphoton microscope based on a commercial laser beam scanning unit (Bio-Rad MRC 1024) and an inverted microscope (Olympus, IX-70). Trains of laser pulses at 80MHz and ~100fs duration were generated by a mode-locked Ti:Sapphire laser (Spectra-Physics Tsunami), which was pumped by a 5W diode laser (Spectra-Physics Millennia). Intensity and polarization were controlled by a Pockels cell (Conoptics 350–50) and a Berek compensator (New Focus 5540). The beam was focused onto the sample by an Olympus UApo/340 20X/NA 0.7 water immersion objective, which also collected the epi-fluorescence. The transmitted SHG was collected by an Olympus XLUMPlanFl 20X/NA 0.95 dipping objective. The average power after the objective was ~70–130mW. For imaging, samples were excited at 774nm and signals were detected with bi-alkali photomultiplier tubes (Hamamatsu HC125-02). SHG was collected behind a focusing lens, an IR-blocking short-pass dichroic and a narrowband emission filter, centered at 387nm (Semrock FF720 and FF01-387/11). Autofluorescence was collected from 400–550nm behind a blue glass filter (Chroma BGG22). Emission spectra were obtained using a liquid nitrogen-cooled CCD spectrometer (Jobin Yvon Spex270M). The spectrometer was coupled to the microscope via an optical fiber replacing the usual bi-alkali photomultiplier in the transmission-direction. To collect one spectrum, a small square area ~50 μm wide was continuously scanned at high zoom for 10 seconds. To reduce photodamage, excitation wavelength was set at 830nm and average power was reduced to ~25mW. Dark counts were subtracted by acquiring a blank spectrum with shutter closed. The spectrometer was calibrated with collagen SHG spectra collected from a rat tendon sample. 2.2.

Tissue preparation

Acute brain slices were prepared from 12 mice, including one with APP Swedish mutation [ 14 ] (obtained from G. K. Gouras), two with APPSwe/PS1 double mutations [ 15 ] (ordered from Jackson Laboratory), two with APPSwe/TauJNPL3 double mutations [ 16 ] (obtained from K. Duff), and three with APPSwe/PS1/Tau triple mutations [ 17 ] (obtained from G. K. Gouras), and four wild-type mice. All mice were at least 1-year old. All preparations were performed in accordance with Cornell University animal use regulations (IACUC protocol 00-46-03). To prepare acute slices, we removed the brain following CO 2 euthanasia. Immediately, the brain was dipped in iced artificial cerebrospinal fluid (ACSF) composed of (in mM): NaCl, 120; KCl, 2.5; NaH2PO4, 1; MgSO4, 1.3, NaHCO3, 25; D-glucose, 10; CaCl2, 2.5, that was saturated with 95% O 2 and 5% CO 2 . Transverse hippocampal slices 300–400μm thick were cut using a vibratome (Campden Instruments). Slices were then incubated in oxygenated ACSF at 35°C for 1 hour. During imaging, slices were held under nylon grid anchors in a flow chamber (Warner Instruments) and perfused with oxygenated ACSF at room temperature. After imaging the intrinsic emissions from unstained brain slices, AD pathology was verified by Thioflavin-S or BTA-1 staining of senile plaques. Brain slices were fixed in 4% paraformaldehyde in PBS in the refrigerator overnight. Next the slices were incubated for 1–2 hours in Thioflavin-S (Sigma, 0.0005–0.001% by weight in PBS), or 40μM BTA-1 (Sigma, 40mM in DMSO then diluted 1:1000 in PBS), and then rinsed three times in PBS. During imaging, the slices were placed in 35mm glass-bottom dishes (Warner Instruments).