The synthesis of the immediate material (2,2-difluoro-1,3-dioxaboryl-pentadione) was performed using a modified procedure ⁷. The immediate compound (0.15g, 0.1mmol) was dissolved in acetonitrile (3.0 ml), and acetic acid (0.2ml), tetrahydroisoquinoline (0.04mL, 0.3mmol), and 6-N,N′-diethyl-3- pyridylaldehyde (0.36g, 2.0mmol) were added. The resulting solution was stirred at 60°C overnight. A black residue was obtained after removing the solvent, and was subjected to flash column chromatography with methylene chloride to produce a black powder (63.0mg, yield: 15.0%). ¹H and ¹³C NMR spectra were recorded at 500 MHz and 125 MHz respectively, and reported in ppm downfield from tetramethylsilane.¹H NMR (CDCl₃) δ(ppm) 1.24 (t, 12H, J = 7.2 Hz), 3.58 (q, 8H, J = 7.2, 14.0 Hz), 5.89 (s, 1H), 6.42 (d, 2H, J = 16.0 Hz), 6.51 (d, 2H, J = 9.0 Hz), 7.66 (dd, 2H, J = 2.4, 9.0 Hz), 7.92 (d, 2H, J = 16.0 Hz), 8.34 (d, 2H, J = 5.0 Hz); ¹³C NMR (CDCl₃) δ(ppm) 18.8, 42.7, 101.6, 106.3, 115.9, 118.6, 136.0, 144.2, 153.4, 159.8, 178.2.

A 1.0ml PBS solution of Aβ42 (250 nM) was added to each well of a 12-well plate, followed by the addition of a DMSO solution (10 μL) of CRANAD-3. The final concentrations of CRANAD-3 added were 50, 100, 250, 500, 750, and 1000 nM, with samples prepared in quadruplicate. The resulting plate was subjected to imaging using IVIS®Spectrum by setting Ex = 530nm, Em = 580nm to 840nm with 14 filters, subject height = 0.2cm, and FOV = C. Spectral unmixing was performed with LivingImage^® 3.2 software by setting Component = 3 (representing 3 fluorophores that include autofluorescence, bound CRANAD-3 and free CRANAD-3), Photo Mask (this tool is for selecting the area of interesting), and Auto constraints (this tool is for constraining parameters to allow user zeroing low band pass and making non-negative contribution from fluorophores. In our experiments, we used the auto default model from the imaging system).

Wild type (20-month old) or APP/PS1 (14-month old) mouse brain tissue was cut into 25-micron slices, fixed in 4 % formalin for 5 min and washed twice with PBS buffer. The tissue was incubated with 0.01% CRANAD-3 (in 50% ethanol) and washed. The tissue was subjected to imaging using the same parameter settings as were used for the phantom imaging.

A 24-month APP/PS1 mouse was intravenously injected with 1.5mg/kg CRANAD-3 (freshly prepared with 15% DMSO, 15% cremophor and 70% PBS buffer). After 1 hour, the mouse was sacrificed and the brain, heart, and spinal cord were collected. The three tissue/organs were subjected to imaging with Ex = 530nm, Em = 580 – 840nm, small binning, f = 2, FOV = B, and exposure time = 0.5s. Spectral unmixing was performed using LivingImage^® 3.2 software by setting component = 3, Photo Mask, and Auto constraints.

To test whether the spectral unmixing technique was capable of differentiating the binding status of probe in brain tissue, we stained a brain slice from a 20-month-old wild type mouse with no Aβ pathology with CRANAD-3 and then imaged the slice using similar imaging parameters as the phantom imaging. In raw images of the slice, we found that the signal from the corpus callosum, the largest white matter structure of the brain, was slightly higher than in other regions (Fig. 2A), suggesting that CRANAD-3 had some interaction with white matter. To test whether the unmixed spectrum for white matter was different from the spectrum of CRANAD-3 bound to Aβ determined from phantom studies, we unmixed the raw images into three components, representing autofluorescence, white matter binding, and free probe spectra. After unmixing, we found that the peak for white matter binding was around 600 nm, a 40 nm difference from that of the Aβ bound spectrum from phantom studies (Fig. 2B and Fig.1C). This was not surprising given that fluorescence probes are known to display a higher intensity and blue shift upon interaction with hydrophobic environments ¹⁵ like the environment of white matter tissue. We found that the peak for the free probe (680nm) was 20 nm shorter than that of the phantom (700nm), a finding likely due to the differences in environment between phantom solution and brain slices (Fig. 2B). The distribution map likewise showed that the CRANAD-3 bound to white matter was mostly present in the corpus callosum area (Fig. 2C #2 and #4), while the free probe was distributed randomly across the tissue (Fig. 2C #3). Our results suggest that spectral unmixing coupled with wavelength responsive probe CRANAD-3 allows us to spectrally and spatially differentiate white matter binding from free probe while providing evidence that binding to non- Aβ structures can be differentiated from Aβ binding by a spectral peak shift.

APP/PS1 mice, a widely used transgenic strain for AD research, express chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and mutant human presenilin 1 protein (PS1-dE9). The “humanized” Mo/HuAPP695swe transgene allows the mice to secrete a human Aβ peptide, leading to a gradual increase Aβ loading during the aging process ^16,17. In our next experiment we used brain slices of a 14-month APP/PS1 transgenic mouse obtained after intravenous injection of CRANAD-3. Interestingly, the raw images of an ex vivo slice showed weak white matter binding (Fig. 3A), indicating that at the time of imaging (2 hours post-injection) most CRANAD-3 had washed out of the white matter. As expected, we obtained well-resolved spectra and images after performing the unmixing procedure (Fig. 3B, C) corresponding to spectral signals of autofluorescence, Aβ bound probe and free probe. From Fig. 3B, it can be seen that the bound probe primarily distributed in cortex area and hippocampus, and this distribution pattern was similar to the Aβ plaques deposit pattern observed from fluorescent microscopic images (SI Fig.3). The spectrum peak of bound probe was 640 nm, the same as the peak obtained in phantom imaging studies (Fig. 1C). Similarly, the spectrum of free probe was very similar to that of in vitro CRANAD-3 stained wild type brain slice (Fig. 2B).

After we demonstrated that the spectral unmixing technique was feasible for brain tissue slices using our CRANAD-3 probe, we tested this technique on the whole brain ex vivo. To this end, we obtained the non-perfused whole brain from a 24-month old APP/PS1 mouse 1 hour after intravenous injection of CRANAD-3. The brain, heart and a portion of spinal cord were imaged as described previously. After unmixing of the raw images, well-resolved spectra were generated for autofluorescence, bound and unbound CRANAD-3 (Fig. 4A). The spectra were similar to those obtained after AD tissue imaging. Remarkably, the unmixed images clearly showed that the bound probe was primarily located in the cortex (Fig. 4B #2), while the unbound probe was present in the blood vessels, across the whole brain and in the heart (Fig. 4B #3). The composite image, the merged/summated image of the images of the unmixed components, indicated that the probe in the blood vessel and the heart primarily existed in its unbound form (red) (Fig. 4B #4), whereas the probe in the cortical regions was bound (blue). Additionally, we noticed that the probe in the spinal cord was mainly present as a bound form (blue) while the blood in the narrow tissue area surrounding the spinal cord contained an unbound probe (red). These unmixed images reflected the fact that Aβ species were primarily present in the cortex and cerebrospinal fluid (CSF) and that their concentration in the blood was considerably lower. In addition, we did not observe the spectral peak for white matter binding. Several things could account for this, including the deeper location of the white matter, washing out of the probe from the white matter, or the embedding of the white matter binding signal into the spectrum of autofluorescence.

The intrinsic difference between wild-type and transgenic AD mice is the absence of Aβ species in the wild-type ^16,17. We reasoned that free probe would be a dominant component in wild-type mice, while probe bound to Aβ species would predominate in APP/PS1 mice. To test this hypothesis, we intravenously injected both wild-type and APP/PS1 mice with CRANAD-3, and imaged them with excitation at 570 nm and emission ranging from 620–840 nm. We selected the 570 nm filter excitation for in vivo imaging because of its better tissue penetration than 535 nm, the excitation filter for ex vivo imaging. We unmixed the raw images using autofluorescence and unbound free probe as two expected major components in wild-type mouse. There was a peak in the unmixed spectra around 670 nm (Fig. 5B) corresponding to the free probe existing in an unbound form. The unmixed spectrum for APP/PS1 mouse was very similar to the spectrum of Aβ bound probe, suggesting that the major component existed in the bound form (Fig. 5A). This result indicates that we could use the spectral unmixing technique to differentiate wild-type from transgenic AD mice.