Miriam Unger

3D spectral imaging with synchrotron Fourier transform infrared spectro-microtomography

We report Fourier transform infrared spectro-microtomography, a nondestructive three-dimensional imaging approach that reveals the distribution of distinctive chemical compositions throughout an intact biological or materials sample. The method combines mid-infrared absorption contrast with computed tomographic data acquisition and reconstruction to enhance chemical and morphological localization by determining a complete infrared spectrum for every voxel (millions of spectra determined per sample).

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

Synchrotron FTIR reveals lipid around and within amyloid plaques in transgenic mice and Alzheimer's disease brain

While the basis of neuronal degeneration in Alzheimers disease (AD) continues to be debated, the amyloid cascade hypothesis remains central. Amyloid plaques are a required pathological marker for post mortem diagnosis, and Aβ peptide is regarded by most as a critical trigger at the very least. We present spectrochemical image analysis of brain tissue sections obtained with the mid-infrared beamline IRENI (InfraRed ENvironmental Imaging, Synchrotron Radiation Center, U Wisconsin-Madison), where the pixel resolution of 0.54 × 0.54 µm(2) permits analysis at sub-cellular dimensions. Spectrochemical images of dense core plaque found in hippocampus and cortex sections of two transgenic mouse models of AD (TgCRND8 and 3×Tg) are compared with plaque images from a 91 year old apoE43 human AD case. Spectral analysis was done in conjunction with histochemical stains of serial sections. A lipid membrane-like spectral signature surrounded and infiltrated the dense core plaques in all cases. Remarkable compositional similarities in early stage plaques suggest similar routes to plaque formation, regardless of genetic predisposition or mammalian origin.

Variable-temperature Fourier transform infrared spectroscopic investigations of poly(3-hydroxyalkanoates) and perturbation-correlation moving-window two-dimensional correlation analysis. Part I: Study of non-annealed and annealed poly(3-hydroxybutyrate) homopolymer.

Generalized two-dimensional correlation spectroscopy (2DCOS) and perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy were applied to explore the melting behavior of non-annealed and annealed poly(3-hydroxybutyrate) (PHB) homopolymer as studied by variable-temperature Fourier transform infrared (FT-IR) spectroscopy. The absorption band of the C=O stretching vibration was employed to investigate the structural changes during the heating process (30–200 °C). Non-annealed PHB showed a recrystallization process in the temperature range 30–120 °C. In the asynchronous 2D correlation spectrum we clearly captured the existence of two components in the crystallinity-sensitive wing of the C=O stretching mode: a well-ordered crystalline state at lower wavenumbers (1718 cm−1) and a less ordered crystalline state at higher wavenumbers (1724 cm−1). These crystallinity-sensitive bands at 1718 and 1724 cm−1, which are not readily detectable in the one-dimensional (1D) FT-IR spectra, share asynchronous cross-peaks with bands at around 1737 and 1747 cm−1 assignable to the C=O stretching absorptions due to the amorphous components. In the case of the melting process of non-annealed PHB in the temperature range 120–200 °C, it is helpful to use the PCMW2D correlation analysis, which indicates the recrystallization between 40 and 110 °C by the shift of the C=O stretching band from 1726 cm−1 to 1722 cm−1 and the sharp change to the broad amorphous C=O stretching absorption at 1747 cm−1 at the melting temperature of PHB around 190 °C. For an annealed sample of PHB only the melting behavior was observed in the PCMW2D correlation analysis by the sharp transition from the crystalline to the amorphous C=O stretching band.

Variable-Temperature Fourier Transform Infrared Spectroscopic Investigations of Poly(3-Hydroxyalkanoates) and Perturbation-Correlation Moving-Window Two-Dimensional Correlation Analysis. Part II: Study of Poly(ε-Caprolactone) Homopolymer and a Poly(3-Hydroxybutyrate)—Poly(ε-Caprolactone) Blend

In the present study, variable-temperature Fourier transform infrared (FT-IR) spectra of a poly(ε-caprolactone) (PCL) homopolymer and a poly(3-hydroxybutyrate) (PHB)-poly(ε-caprolactone) (PCL) blend were analyzed by generalized two-dimensional correlation spectroscopy (2DCOS) and perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy. The C=O stretching vibration bands of PCL and PHB were employed to explore the structural changes in the PCL homopolymer and the PHB–PCL blend during the heating process. For the melting of PCL homopolymer in the temperature range of 50 to 70 °C, we observed in the synchronous and asynchronous 2D correlation spectra one crystalline (1724 cm−1) and one amorphous (1737 cm−1) C=O stretching vibration band, which are also detectable in the one-dimensional FT-IR spectra. This result was also confirmed by PCMW2D correlation spectroscopy. During the heating process of the PHB–PCL blend in the temperature range 30–200 °C, the PCMW2D correlation analysis provided detailed information. Thus, in the synchronous PCMW2D correlation spectrum the melting of PCL was observed in the temperature region between 30 and 70 °C. The recrystallization of PHB in the blend in the temperature range 70–120 °C was accompanied by a shift of the C=O stretching band from higher wavenumber (1724 cm−1) corresponding to an imperfect crystalline state to the lower wavenumber (1721 cm−1) characteristic of a well-ordered crystalline state. In the temperature range 120–200 °C the melting process of PHB in the blend is captured by a sharp transition from the crystalline (1722 cm−1) to the amorphous (1747 cm−1) C=O stretching band.

In Situ Orientation Studies of a Poly(3-hydroxybutyrate)/Poly(e-caprolactone) Blend by Rheo-Optical Fourier Transform Infrared Spectroscopy and Two-Dimensional Correlation Spectroscopic Analysis

In the present study, the orientation of a poly(3-hydroxybutyrate) (PHB)/poly(ε-caprolactone) (PCL) blend was monitored during uniaxial elongation by rheo-optical Fourier transform infrared (FT-IR) spectroscopy and analyzed by generalized two-dimensional correlation spectroscopy (2D-COS). The dichroism of the δ(CH2) absorption bands of PHB and PCL was employed to determine the polymer chain orientation in the PHB/PCL blend during the elongation up to 267% strain. From the PHB and PCL specific orientation functions it was derived that the PCL chains orient into the drawing direction while the PHB chains orient predominantly perpendicular to the applied strain. To extract more detailed information about the polymer orientation during uniaxial elongation, 2D-COS analysis was employed for the dichroic difference of the polarization spectra recorded during the drawing process. In the corresponding synchronous and asynchronous 2D correlation plots, absorption bands characteristic of the crystalline and amorphous regions of PHB and PCL were separated. Furthermore, the 2D-COS analysis revealed that during the mechanical treatment the PCL domains orient before the PHB domains.

Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells

Advancements in widefield infrared spectromicroscopy have recently been demonstrated following the commissioning of IRENI (InfraRed ENvironmental Imaging), a Fourier Transform infrared (FTIR) chemical imaging beamline at the Synchrotron Radiation Center. The present study demonstrates the effects of magnification, spatial oversampling, spectral pre-processing and deconvolution, focusing on the intracellular detection and distribution of an exogenous metal tris-carbonyl derivative 1 in a single MDA-MB-231 breast cancer cell. We demonstrate here that spatial oversampling for synchrotron-based infrared imaging is critical to obtain accurate diffraction-limited images at all wavelengths simultaneously. Resolution criteria and results from raw and deconvoluted images for two Schwarzschild objectives (36×, NA 0.5 and 74×, NA 0.65) are compared to each other and to prior reports for raster-scanned, confocal microscopes. The resolution of the imaging data can be improved by deconvolving the instrumental broadening that is determined with the measured PSFs, which is implemented with GPU programming architecture for fast hyperspectral processing. High definition, rapidly acquired, FTIR chemical images of respective spectral signatures of the cell 1 and shows that 1 is localized next to the phosphate- and Amide-rich regions, in agreement with previous infrared and luminescence studies. The infrared image contrast, localization and definition are improved after applying proven spectral pre-processing (principal component analysis based noise reduction and RMie scattering correction algorithms) to individual pixel spectra in the hyperspectral cube.

Molecular Weight Dependence of the Thermal Degradation of Poly(e-caprolactone): A Thermogravimetric Differential Thermal Fourier Transform Infrared Spectroscopy Study

The effect of molecular weight on the thermal degradation of poly(ε-caprolactone) (PCL) was investigated by thermogravimetric analysis in combination with differential thermal analysis and Fourier transform infrared spectroscopy (TGA/DTA/FT-IR). The measurements were made in the temperature range 40–720 °C and it was found that PCL undergoes completely different degradation processes in nitrogen and oxygen atmosphere. Thus, in nitrogen atmosphere low molecular weight (Mn = 10000 g/mol) PCL (PCL10k) decomposed in a three-step mechanism. The evolved gases detected by FT-IR spectroscopy were identified as ε-caprolactone, 5-hexenoic acid, CO2, and methyl pentanoate and traces of H2O. In the case of high molecular weight (Mn = 80 000 g/mol) PCL (PCL80k) only a two-step degradation was observed. By FT-IR spectroscopy 5-hexenoic acid, CO2, H2O, and methyl pentanoate were detected as decomposition products. In an oxygen environment, similar degradation products were detected for the different molecular-weight PCLs. The recorded FT-IR spectra of the evolved gases were identified as CO2, CO, H2O, and short-chain carboxylic acids.

Multi-beam Synchrotron FTIR Chemical Imaging: Impacts of Schwarzschild Objective and Spatial Oversampling on Spatial Resolution

IRENI (InfraRed ENvironmental Imaging) is a recently commissioned FTIR chemical imaging beamline at the Synchrotron Radiation Center in Madison, WI. This novel beamline extracts 320 mrads of radiation, horizontally, from one bending magnet. The optical transport separates and recombines the beam into 12 parallel, collimated beams to illuminate a commercial FTIR microspectrometer (Bruker Hyperion 3000) that is apertureless and equipped with a multiple element detector. The beams are partially overlapped and defocused, similar to widefield microscopy, homogeneously illuminating a relatively large sample area compared to single beam arrangements. The effective geometric size of a pixel at the sample plane is defined by the magnification of the optics, and as expected, objectives with varying magnification and numerical apertures (NAs) impact the diffraction-limited resolution that can be obtained in the resulting images. We demonstrate here that spatial oversampling for synchrotron-based infrared imaging is critical to obtain diffraction-limited images at all wavelengths simultaneously. In this article measured and simulated point spread functions (PSF), resolution criteria and results from raw and deconvoluted images for two common Schwarzschild objectives (36x, NA 0.5 and 74x, NA.65) are compared to each other and to prior reports for confocal microscopes. The resolution of the imaging data can be improved by deconvolving the instrumental broadening that is simulated with the PSF. The contrast resolution for IRENI without employing apertures is similar to dual aperture, confocal microscopes.

Water uptake of poly(2-N-alkyl-2-oxazoline)s: temperature-dependent Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation analysis (2DCOS)

A library of poly(2-oxazoline)s with varying length of the alkyl side-chain has been investigated by variable-temperature Fourier transform infrared (FT-IR) spectroscopy. These polymers are suitable for studies of structure-property relationships as their cationic ring-opening polymerization and the relatively facile monomer synthesis enable a control of the molecular structure. In this contribution, the number of carbon atoms in the linear side-chain is systematically varied from a short methyl to a long nonyl group. Previous studies showed that the sample library can be split in two groups: poly(2-oxazoline)s with a short side-chain (methyl-, ethyl-, and isopropyl-) exhibit hygroscopic behavior, while those with longer side-groups (butyl- and longer) were found to be semi-crystalline. To gain further insight into the mechanisms of hydrogen bonding and crystallization, temperature-dependent infrared (IR) spectroscopy has been applied in the current study. The processes involved have been monitored by generalized two-dimensional correlation spectroscopy (2DCOS) and perturbation-correlation moving-window two-dimensional correlation spectroscopy (PCMW2D) in the C=O stretching region around 1645 cm−1. These advanced analysis techniques provided valuable additional information on the material behavior during heating. As water is removed from the samples in the course of the heating process, it was possible to clearly distinguish between “loosely associated” and hydrogen-bonded water. Furthermore, the melting process of the semi-crystalline samples could be depicted. For the poly(2-isopropyl-2-oxazoline) even a crystallization process could be monitored in the temperature range between glass transition and melting.