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FT-IR microspectroscopy available for the first users

FT-IR microspectroscopy available for the first users

The Hyperion 3000 FT-IR microscope has been completed with the last component, the Focal Plane Array detector (128x128 pixels), and is now available for test measurements for users. Ultimately, the microscope, when coupled to the synchrotron beam, will be one of the two end stations of the CIRI beamline. Future users are encouraged to contact the beamline team for test measurements.

Hyperion 3000 and potential applications

The CIRI (Chemical InfraRed Imaging Beamline) beamline, which enables the use of infrared radiation over a wide spectral range for research, will have two end stations - an FT-IR imaging microscope and an AFM-IR/s-SNOM microscope. Although the beamline is currently under construction, with commissioning scheduled for 2023, the possibility of using one of the end stations has already emerged. A Hyperion 3000 FT-IR microscope with a Vertex 80v spectrometer (Bruker Optics) has been installed in the research laboratory close to the CIRI beamline, with an extremely wide range of applications, starting from materials research, polymers, chemical compounds, forensics to art conservation or life sciences. Thanks to the microscope's modular design, it can be adapted to the specific requirements of each experiment.

Head of the FT-IR beamline, Tomasz Wróbel, PhD, speaks about the microscope's applications as follows: "FT-IR microspectroscopy makes it possible to simultaneously analyze the chemical composition and structure of a sample on a micrometer scale, which greatly facilitates and accelerates advanced research in many scientific fields. One possible application of such a microscope is to make measurements of whole biopsies of normal and cancerous tissues for further chemometric analysis and classification."

The first test measurements of biopsies, other samples of biological origin and polymers on the microscope have been carried out by the beamline's research team, and the results are very promising.

It is one of four microscopes of this class in Poland. In addition, the synchrotron source will provide high brightness infrared radiation covering the range from near to far infrared, making FT-IR microspectroscopy with a synchrotron source an exceptional research instrument in the world. The high quality of the spectrum is ensured by an excellent signal-to-noise ratio.

Karolina Kosowska, Ph.D., a materials engineer from CIRI's beamline, talks about the microscope's applications in art department research, among others: "Resolution at the level of a few micrometers makes it possible to geochemically analyze the organic and inorganic matter in shale, or to visualize the distribution and simultaneous chemical analysis of the pigments used by painters to create a painting several hundred years ago."

Scientists interested in the possibilities of using the FT-IR microscope in their research and performing research at the SOLARIS Centre are invited to contact the beamline's team. 

Below are some examples of publications based on FT-IR spectroscopy. 

1. FT-IR microspectroscopy and chemometric analysis for classification of tissue types in biopsies

"Translation of an esophagus histopathological FT-IR imaging model to a fast quantum cascade laser modality." - https://doi.org/10.1002/jbio.202000122

cancerous and healthy biopsies predicted images

 

Figure 1. Results of Random Forest approach prediction of three tissue classes: mature epithelium, cancerous epithelium and others, for esophagus dewaxed TMA measured with FT-IR system in transmission mode (left figure panel). Single cancerous and healthy biopsies predicted images with corresponding H&E stained biopsies images (right panels).

 

 

To cut the theoretical basis of vibrational spectroscopy short, we can say that oscillations of chemical bonds are able to absorb IR light at well-defined wavelengths. Spectroscopic spectra specific for each molecule allow for the chemical identification at a given measuring point and even the architecture of the material. 

schemat występowania pasm do przykładowych wiązań

 

 

 

Figure 2: Infrared spectroscopy correlation table. From: M.J. Jafari „Application of Vibrational Spectroscopy in Organic Electronics” 2017.

 

2. Orientation of macromolecules in a biopsy

Macromolecular Orientation in Biological Tissues Using a Four-Polarization Method in FT-IR Imaging - doi: 10.1021/acs.analchem.0c02591

Spatially resolved macromolecular orientation in biological tissues using FT-IR imaging - https://doi.org/10.1016/j.clispe.2021.100013

Advanced methods of imaging larger areas of the sample using, among other things, linearly polarized light, make it possible to determine the orientation of molecules in two- and even three-dimensional space. The given publications presented the possibility of using the "four polarization" method to determine the orientation of collagen fibers in biopsies and the organization of macromolecules in polymer spherulites in three-dimensional space.

Igłowa biopsja trzustki

 

Figure 3. Histological H&E stained image of biopsy, Hermans' orientation function results calculated for amide III, the orientation of amide I, and amide III bands represented by the azimuthal angle.

 

 

 

 

 

 

 

 

 

3. 3D imaging of the orientation of molecules in the polymer

Super-resolved 3D mapping of molecular orientation with vibrational techniques - doi: 10.26434/chemrxiv-2021-1hd81-v3

Cząsteczka polikaprolaktamu

 

 

Figure 4: 3D space orientation of vibrations in a polycaprolactam molecule. Dragging νas(C-O-C) vibration is parallel to the main chain of the molecule, ν(C=O) is perpendicular.

 

 

4. Mapping and imaging of samples

Mapping and imaging methods allow sample visualization using successive wavelengths with a selected interval (spectral resolution). As a result, we collected a cube of three-dimensional data with two spatial and one spectral dimension. The image panes are organized in a stack as a function of wavenumbers. Vibrational spectroscopic mapping and imaging reveal trends in a sample, which would be hard using single-point measurement.

Schematic of a three-dimensional spectral data matrix

 

Figure 5: Schematic of a three-dimensional spectral data matrix.

 

 

 

 

 

 

 

 

 

 

Mapping involves the successive collection of successive points along a designated line or two-dimensional grid of points. Normally, a mercury-cadmium telluride MCT detector is used for signal collection, and the area of interest is narrowed by an aperture. The imaging method allows the simultaneous collection of multiple spectra from different points using a focal plane array (FPA) detector. The use of a detector array significantly reduces measurement time. In the case of a 128x128 detector, information from more than sixteen thousand points is collected at the same time. The pixel size, which should not be confused with resolution, depends on the total magnification and physical size of the detector.

At the first #CIRI end-of-line station (former name: SOLAIR), users have the ability to image various types of samples with pixel sizes as small as 1.1 μm.

 

Written by Karolina Kosowska, Kinga Wróbel, Tomasz Wróbel

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