Five micrometer deparaffinized sections of decalcified lumbar disc tissues were collected from vehicle and D + Q 14–23 M cohort group (n = 3 disc/animal, 6 animals/group) and used to acquire FTIR spectral imaging data using methods previously described40 (link). Data was collected using Spectrum Spotlight 400 FTIR Imaging system (Perkin Elmer, Shelton, CT), operating in the mid-IR region of 4,000 - 850 cm−1 at a spectral resolution of 8 cm−1 and spatial resolution of 25 μm. Spectra were collected across the mid-IR region of three consecutive sections/disc to minimize section-based variation. Using the ISys Chemical Imaging Analysis software (v. 5.0.0.14) mean second-derivative absorbances in the collagen side-chain vibration (1338 cm−1) regions were quantified and compared in Veh and D + Q AF and NP compartments. The preprocessed spectra were used for K-means cluster analysis to define anatomical regions and tissue types within the tissue section spectral images7 (link), which represent collagen peak. Clustering images were obtain using Spectrum Image Software.
Spectrum spotlight 400 ft ir imaging system
Manufactured by PerkinElmer
Sourced in France
The Spectrum Spotlight 400 FT-IR Imaging System is a Fourier Transform Infrared (FT-IR) imaging spectrometer. It is designed to perform high-resolution infrared imaging and spectroscopic analysis of samples.
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9 protocols using spectrum spotlight 400 ft ir imaging system
1
FTIR Spectral Imaging of Decalcified Lumbar Discs
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Spectral Imaging Analysis of Calcified Discs
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IR spectral imaging data in the mid‐IR region, 4,000–800/cm at 8/cm spectral resolution and 25 μm spatial resolution was acquired from 10‐μm‐thick calcified caudal disc sections from 6M and 23M LG/J mice (n = 3 discs/group) using a Spectrum Spotlight 400 FT‐IR Imaging system (Perkin Elmer, CT) (Choi et al., 2018 ; Mohanty et al., 2019 ). Absorbance for proteins in the amide I region, 1,665/cm; phosphate vibration region, 960 cm−1 and the carbonate at 870/cm were recorded (Berzina‐Cimdina & Borodajenko, 2012 ). The preprocessed spectra were used for K‐means cluster analysis to define anatomical regions and tissue types within the tissue section spectral images (Choi et al., 2018 ), which represent each analysed peak and ratio. clustering images were obtained using Spectrum Image Software.
3
Characterization of Fullerol and 3HFWC
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UV–vis–NIR characterization of both FD-C60 (fullerol) and SD-C60 (3HFWC) was performed using a Lambda 500 spectrometer, Perkin-Elmer, USA, in the range of 250–3000 nm. FTIR characterization of both FD-C60 (fullerol) and SD-C60 (3HFWC) was performed using the Spectrum Spotlight 400 FTIR Imaging System, Perkin-Elmer, Waltham, MA, USA, in the range of 2500–14,000 nm. Additionally, the following instruments were used for the characterization: near-infrared spectrometer Lambda 1050+, Perkin Elmer, up to 2600 nm; Infrared Spectrometer Spectrum Two FT-IR, Perkin Elmer; Laser HeNe Class 1, with a scanned range of 370–7800 cm−1; UV–vis spectrometer Specord 205, Analytik Jena (Jena, Germany), with a resolution of ±0.5 nm and a scanned range of 190–1100 nm.
4
Infrared Microspectroscopy of Microcalcifications
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Microcalcifications were characterized using Fourier transform infrared microspectroscopy (µ‐FTIR). Tissue sections (4‐µm) were deposited on low‐emission microscope slides (MirrIR, Keveley Technologies, Tienta Sciences, Indianapolis). FTIR hyperspectral images were recorded with a Spectrum spotlight 400 FTIR imaging system (Perkin Elmer Life Sciences, Courtaboeuf, France), with a spatial resolution of 6.25 micrometre and a spectral resolution of 8 cm‐1. Each spectral image covering a substantial part of the tissue consisted of about 30,000 spectra.
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Infrared Spectroscopy of Decalcified Discs
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FTIR Imaging of Plant Samples
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Once the plants were 7 weeks old, the samples were dehydrated in an incubator at 60°C for 75 minutes and allowed to cool to room temperature. Under these conditions, the slides were observed to dry uniformly within 15 minutes; however, an additional hour of gentle drying was used to drive off any residual moisture. Slides were stored in a dark, desiccated environment until FTIRI was performed. All FTIR images were obtained from the dried samples using a Spectrum Spotlight 400 FTIR Imaging System (PerkinElmer) in transflectance mode. Spectra were collected in the mid-IR region from 4000 to 750 cm -1 using a 16 pixel MCT (Mercury Cadmium Telluride) array detector with a 25 µm pixel size. The spectral resolution was 8 cm -1 and 16 scans per spectrum were collected.
FTIR spectra were also collected from the individual components of the media, i.e. sucrose, WPM, Phytagel, and ammonium nitrate. Spectra were collected in the mid-IR region from 4000 -750 cm -1 using a spectral resolution of 4 cm -1 and 8 scans per spectrum. Spectra of the individual components were collected using Beamline U2B at the National Synchrotron Light Source at Brookhaven National Laboratory.
FTIR spectra were also collected from the individual components of the media, i.e. sucrose, WPM, Phytagel, and ammonium nitrate. Spectra were collected in the mid-IR region from 4000 -750 cm -1 using a spectral resolution of 4 cm -1 and 8 scans per spectrum. Spectra of the individual components were collected using Beamline U2B at the National Synchrotron Light Source at Brookhaven National Laboratory.
7
Microcalcification Characterization by μ-FTIR
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Microcalcifications were characterized using Fourier transform infrared micro-spectroscopy (µ-FTIR). Tissue sections (4 µm) were deposited on low-emission microscope slides (MirrIR, Keveley Technologies, Tienta Sciences, Indianapolis). FTIR analysis was performed in serial sections adjacent to tissue sections stained with Yasue technique. FTIR hyperspectral images were recorded with a Spectrum spotlight 400 FT-IR imaging system (Perkin Elmer Life Sciences, Courtaboeuf, France), with a spatial resolution of 6.25 micrometre and a spectral resolution of 8 cm -1 . The spectra were recorded in the 4000-700 cm -1 mid-infrared range. Each spectral image covering a substantial part of the tissue consisted of about 30,000 spectra.
8
FT-IR Imaging of Microcalcifications
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Microcalcification phases were characterized using µFourier Transform InfraRed spectrometry. Four micrometer tissue sections were deposited on low emission microscope slides (MirrIR, Keveley Technologies, Tienta Sciences, Indianapolis). FT-IR hyperspectral images were recorded with a Spectrum spotlight 400 FT-IR imaging system (Perkin Elmer Life Sciences, France), with a spatial resolution of 6.25 µm and a spectral resolution of 8 cm - 1 . The spectra were recorded in the 4000-700 cm -1 mid-InfraRed range. Each spectral image, covering a substantial part of the tissue, consisted of about 30,000 spectra.
9
Ultrastructural and Spectroscopic Analysis of Renal Papilla
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Right renal papilla were fixed in 2.5% glutaraldehyde in 0.1 mmol/L cacodylate buffer (pH 7.4) at 4 C. Fragments were fixed in 1% osmium tetroxide, dehydrated using alcohol series, and then embedded in epoxy resin. Semithin sections (0.5 mm) were stained using toluidine blue. Ultrastructure sections (80 nm) were contrast-enhanced using UranyLess staining (Delta Microscopies, Mauressac, France). A JEOL 1010 electron microscope (JEOL, Ltd., Tokyo, Japan) with a MegaView III camera (Olympus Soft Imaging Systems GmbH, Munster, Germany) was used to analyze tissues.
mFourier Transform InfraRed Spectroscopy Microcalcifications were characterized using m-Fourier transform infrared (FT-IR) spectrometry on 4-mm tissue sections deposited on low-emission microscope slides (MirrIR; Keveley Technologies, Tienta Sciences, Indianapolis, IN). FT-IR hyperspectral images were analyzed with a Spectrum spotlight 400 FT-IR imaging system (PerkinElmer), at a spatial resolution of 6.25 mm and a spectral resolution of 8 cm À1 . The spectra were recorded in the 4000 to 700 cm À1 mid-infrared range.
mFourier Transform InfraRed Spectroscopy Microcalcifications were characterized using m-Fourier transform infrared (FT-IR) spectrometry on 4-mm tissue sections deposited on low-emission microscope slides (MirrIR; Keveley Technologies, Tienta Sciences, Indianapolis, IN). FT-IR hyperspectral images were analyzed with a Spectrum spotlight 400 FT-IR imaging system (PerkinElmer), at a spatial resolution of 6.25 mm and a spectral resolution of 8 cm À1 . The spectra were recorded in the 4000 to 700 cm À1 mid-infrared range.
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