Hyperion 3000 microscope

Spheres made of Poly(methyl methacrylate) (PMMA), with a diameter of $5.5\mathrm{\mu }$ m, were measured using a Vertex 70 FTIR spectrometer coupled with a Hyperion 3000 microscope (both Bruker Optik, Ettlingen, Germany). The measurements were recorded at 15 $\times$ magnification with a numerical aperture of 0.4, using a fully open aperture and a mercury cadmium telluride focal plane array (MCT-FPA) detector. The spheres were placed on 1 mm thick CaF₂ microscope slides, using perfluoronoane as a dispersing agent. Spectra were recorded in the range 899–3844 cm ${}^{-1}$ , with a spectral resolution of 8 cm ${}^{-1}$ and digital spacing of 3.86 cm ${}^{-1}$ . 132 scans were averaged for each spectrum. Two images were collected from each slide, from in total 3 slides. Then spectra from single spheres and double-spheres were selected manually. These spectra were first preprocessed using multiplicative signal correction (MSC) to standardize the spectra with respect to scaling and constant offset, and then averaged.

Fourier Transform Infrared microspectroscopy (μFTIR) based on synchrotron radiation was carried out at the MIRAS beamline of ALBA synchrotron light source (Catalonia, Spain) [16 (link)]. A Hyperion 3000 microscope equipped with a 36× magnification objective and coupled to a Vertex 70 spectrometer (Bruker, Billerica, MA, USA) was used. The spectra collection was performed in transmission mode at 4 cm⁻¹ spectral resolution, 10 μm × 10 μm aperture dimensions and 128 scans. All spectra were obtained by means of Opus 7.5 software (Bruker, Billerica, MA, USA). The measuring range was 4000–600 cm⁻¹ wavenumbers, and zero filling was performed with fast Fourier transform (FFT), so that we obtained one point every 2 cm⁻¹ in the final spectra. Background spectra were collected from a clean area of each CaF₂ window every 10 min. A mercury cadmium telluride (MCT) detector was used, and both the microscope and spectrometer were continuously purged with a flow of dried air. For each studied area, approximately 100 spectra with a step size of 50 μm × 50 μm for human samples and 30 μm × 30 μm for mouse samples were acquired. In order to represent regional differences with high spatial resolution in the tissue, maps of 200 spectra with a step size of 6 μm × 6 μm were performed in one representative sample of each species containing GM and WM areas.

The infrared spectra were acquired with a Fourier Transform Infrared (FT-IR) spectrometer Vertex 70 v (Bruker, Leipzig, Germany) using the MCT mid-band detector D316025. The spectra were obtained using a 20× germanium ATR objective mounted on a Hyperion 3000 microscope (Bruker, Leipzig, Germany) by pressing the objective onto the surface of the PET foil which was placed onto the surface of a gold substrate. The contact pressure between the germanium crystal and the surface of the PET foil was >0.5 N (pressure level 2 of the ATR objective). The spectral resolution was 4 cm⁻¹.The absorbance spectra were registered in the range of wavenumber 600–4000 cm⁻¹. The spectra were corrected by subtracting a constant offset equal to the mean-value of the absorbance in the wavenumber range 3150–3200 cm⁻¹. Each spectrum was normalized by dividing it by the maximum absorbance of the first peak located in the wavenumber range 690–790 cm⁻¹. The results are an average of 8 spectra for each sample.

The same cells as analysed by SXT after PFA fixation were measured by SR-μFTIR on the MIRAS beamline at the ALBA Synchrotron using a Hyperion 3000 Microscope equipped with a 36× magnification objective coupled to a Vertex 70 spectrometer (Bruker) (Yousef et al., 2017 ▸ ). The measuring range was 650–4000 cm⁻¹. Spectra collection was carried out in transmission mode at 4 cm⁻¹ resolution with aperture dimensions of 8 × 8 µm and from 128 to 256 scans. Zero filling was performed with the fast Fourier transform so that in the final spectra there was one point every 2 cm⁻¹. Background spectra were collected from an empty area (a region without Quantifoil) every 15 min. An MCT detector with 50 µm resolution, a responsivity at 4.8 mA of 173 550 V W⁻¹ and a field of view (FOV) of 45 µm was used. The microscope and spectrometer were continuously purged with nitrogen gas.

SR-μFTIR was performed at the MIRAS beamline^{35 (link)} at ALBA synchrotron (Catalonia, Spain), using a Hyperion 3000 Microscope that was equipped with a 36× magnification objective coupled to a Vertex 70 spectrometer (Bruker). The measuring range was 650–4000 cm⁻¹ and the spectrum collection was carried out in transmission mode at 4 cm⁻¹ resolution, 10 μm × 10 μm aperture dimensions, and co-added from 64–128 scans. Zero filling was performed with fast Fourier transform (FFT) so that in the final spectra there was one point every 2 cm⁻¹. Background spectra were collected from a clean area of the CaF₂ window every 10 min. Mercury−cadmium−telluride (MCT) detector was used and the microscope and spectrometer were continuously purged with nitrogen gas. For each condition (age) 3 different animals were measured. For each animal, around 1000 spectra were acquired and in the case of APP/PS1 mice 3 to 5 plaques were measured. Areas with no plaques were also measured in APP/PS1 and WT animals (the spectra from areas with no plaques in APP/PS1 animals appear together with the spectra from the WT animals in the PCA analysis).

For both studies, data were acquired in transmission using the × 36 objective/condenser optics on a Hyperion 3000 microscope coupled to a Bruker Vertex 80 FTIR spectrometer at the MIRIAM beamline B22 at DLS, using a liquid nitrogen (LN₂)-cooled mercury-cadmium-telluride (MCT), high-sensitivity 50 μm pitch detector and 15 × 15 μm² slit size at the sample.
Two hundred fifty-six co-added scans (circa 35 s) were used for both background and sample measurements. A dry area of the sample was used for the background scan. A minimum of 65 cells per sample loading were selected for measurement using OPUS 7 software, with a corresponding measurement of the bulk PBS layer taken from a cell-free area adjacent to each selected cell for use in water correction. This gave a minimum of 130 spectra in each measurement and equates to approximately 90 min measuring time for optimal spectral quality.