Applications & sample preparation BMA Lausanne
Which biological information can be obtained by FTIR microspectrometry?
FTIR spectroscopy is dedicated to identifying the chemical composition of samples. The vibrations of different chemical species, such as functional groups of molecules, occur in defined ranges of infrared spectrum. Therefore, there is the possibility of assigning a particular peak in the spectrum to the presence of a specific chemical moiety, in many cases the use of FTIR spectra libraries simplifies this analysis when available. As biological samples can be chemically complex and as there is potential for a large number of interactions between molecules, infrared spectra can be rather complicated. Thus, the analysis usually focus on identifying alterations in the FTIR spectra either between different samples or at different sites within the same sample. The information obtained from FTIR data analysis can be useful in comparison of plants (either at the whole tissue level or as an extract), according to mutations, pathogen attack or chemical treatment.
Detailed description of sample preparations for different FTIR applications / measurement modes:
There are several ways to prepare samples for FTIR analysis. We do our best to get the highest possible quality of IR spectra from any plant material arriving in our lab. The type and the state of the plant material determines the infrared technique used for analysis. Measurement are possible in transmission, reflection or attenuated total reflection (ATR) IR modes. All three type of spectra can be measured either by IR microscopy or by IR spectroscopy. Any kind of plant material (leaves, stems, roots, etc.), which is transparent for infrared radiation, is suitable for all three FTIR microscopy measurement modes. The ATR mode is used to analyze the chemical composition of the surface of a plant material. With this techniques also probes that are non-transparent for IR radiation can be analyzed by FTIR microscopy. In the spectroscopy mode, all three techniques (transmission, reflection or ATR) can be applied to perform chemical analysis of emulsions, liquid or solid samples, obtained at any stage of plant material isolation.
In the case of microscopic measurements, minimal treatment of plant material is desired. Each chemical treatment of a sample could influence the chemical composition of a studied tissue. However, water, which is a dominant component of biological tissues absorbs strongly in the infrared region and its presence is a critical issue. Therefore we work with dried plant material. Before drying, the studied tissue is placed in a chemical bath (methanol, iso-propanol, etc.) to remove chlorophyll or other compounds, which disturb FTIR analysis. In general, there is a separate procedure of sample preparation for each kind of biologic material. A special branch of FTIR microscopic measurements is thin section analysis (microtomy, cryotomy). The best quality of spectra are obtained if the thickness of the samples is in the 5-50 µm range. Please note that polymers used for tissue embedding have a strong IR spectrum, therefore cryotomy is the preferred way of thin section preparation.
Measurements in transmission mode are performed with the help of a IR-transparent KBr disk (Φ 12 mm). Samples are put on one or are placed between two disks. In the case of the reflection mode, aluminium and gold coated plates (25 x 75 mm) or the 12-well alumina plates (Φ 4 mm) are available for sample mounting. The ATR (attenuated total reflection) accessories enable spectra collection for samples placed on dedicated holder.
Details of sample preparation and data acquisition in the FTIR spectroscopy mode:
- Transmission mode: Liquid samples (extracts, solutions) are placed in quartz cuvettes into the spectrometer sample compartment. The solvent, in which analyzed substance is dissolved, determines the thickness of the cuvette. The water solutions are usually measured in thin cuvettes (0.1 mm), CCl4 or chloroform solutions in 1-10 cm ones, depending on the concentration of dissolved chemical species.
Solid samples (powders) are dissolved in dried, powdered KBr, usually in the concentration range of 1-5% per weight. Next, pellets are made using a KBr press. Resulting disks are placed in sample holder in measurement chamber.
- Transmission mode, 96-well plate assembly: Analyzed substance (thin material, powder) is deposited on top of a 96-well silica cell; thin films could be prepared on each well by solvent evaporation from solutions or suspensions.
- ATR mode: Solid samples (surface of plant material, powders, etc.), liquids or emulsions can be measured by direct sample placement on a diamond crystal. There is also a possibility for the preparation of a thin film from solution or suspension directly on the crystal by gentle evaporation the solvent.