Leveraging FTIR Spectrometers in Biological Research

By Rebecca Ingle, Ph.DReviewed by Megan Craig, M.Sc.

Fourier transform infrared (FTIR) spectroscopy is a powerful technique for the quantitative and qualitative analysis of biological systems. In the life sciences, FTIR spectrometers are used for the analysis of both potential therapeutic compounds, as well as studies of the structure and function of biological matter such as proteins.^1,2

Infrared spectroscopy works by detecting which frequencies of infrared radiation are absorbed by the target sample. The frequencies of light absorbed are dictated by the frequencies of various vibrational modes in the target system. As the frequencies of given vibrational modes are dependent on both the nature of the atoms involved in the motion and the local chemical environment around the atoms, the spectrum of IR frequencies provides a characteristic ‘fingerprint’ that can be used for the assignment of a chemical or biological species and potentially quantification of its concentration.

FTIR spectrometers and the hardware for performing IR spectroscopy, therefore, play a crucial role in the life sciences. Biological samples often exhibit high degrees of chemical complexity – made of hundreds or thousands of atoms with various elements and functional groups. Many proteins and other biological species exhibit poor solubility, so a suitable FTIR spectrometer will need a good limit of detection.³

High throughput techniques are commonly used for screening in the biological sciences, so FTIR spectrometers suitable for biological research often face a number of demands in terms of sensitivity, measurement time and limit of detection.

Operating Principles of FTIR Spectrometers

An FTIR spectrometer has a slightly different design from a dispersive IR spectrometer as FTIR instruments typically offer improved scan times, resolution, and signal-to-noise ratios. Most large commercial spectrometer suppliers, such as Shimadzu, Bruker, Thermo Fischer and Perkin Elmer, all offer extensive FTIR ranges.

Edinburgh Instruments and Jasco also offer a number of options as well as extensive application notes showing how FTIR can be applied in a number of research areas.

A dispersive spectrometer normally relies on scanning in the incident wavelength that is focused onto the sample and the overall energy resolution is defined as a function of the incident and exit energy bandwidth. A reference beam, which does not pass through the sample, is used to monitor the beam intensity assuming no absorption and can be used as a correction factor for the true, sample-only absorption.

A FTIR spectrometer has a design that is based on an Interferometer and recording interferograms that must be Fourier Transformed to recover the infrared transmission or absorption spectrum.

The broadband IR beam is split in two with a beamsplitter and each beam passes through an ‘arm’ of the interferometer, one side with a stationary mirror and the other with a moving mirror. Opposite the moving mirror is the sample with a detector behind it. The path length of the experiment is known to good precision and tuned by moving one of the mirrors to record the interferogram.

FTIR Spectrometers in Biological Research

The key advantages of the use of FTIR spectrometers in the life sciences include rapid distinction between species such as fatty acids, carbohydrates, nucleic acids and some lipid and protein structures.⁴ Variations like attenuated total reflectance FTIR (ATR-FTIR) are also very useful for measuring solid pellets like pharmaceuticals and are often used for quality control.⁵

FTIR spectrometers are a mature and robust technology with a number of chemometrics solutions available for data analysis, as well as extensive spectral databases for compound identification. Some recent applications in the biological sciences of high throughput FTIR have been to monitor plant pollination in the environment.⁶

As plant pollen has several unique IR signatures that relate to the plant's phenotype, high throughput semi-automated FTIR spectrometers could be used to screen large numbers of samples over several years to build up an understanding of how pollen was migrating in an entire ecosystem.

The researchers could then understand how the biochemical variation of the plants in the measured regions was evolving as a function of time.

Combining FTIR Spectrometers with Histology

Histology and medical diagnostics have been another highly active area for FTIR research.⁷ FTIR spectrometers are reasonably easy to miniaturize and incorporate into point-of-care devices.

A recent review of many studies on skin cancer diagnostics with FTIR spectrometers has shown that the chemical information provided by FTIR is particularly invaluable in distinguishing between melanoma and non-melanoma skin cancers and evaluating the metastatic potential of tumor cells, which can be used to predict disease progression.

Combining FTIR spectrometers with nanoimaging capabilities has been one major development for the medical in life sciences as being able to spatially resolve the chemical information that can be obtained with FTIR allows for very detailed analysis of biological tissues and structures.⁸

Researchers have been using FTIR spectrometers to understand how carbonate-forming marine organisms create their shells and the local variations in the chemical composition of the shells. FTIR could distinguish not just the chemical composition but also differences in the atomic lattice arrangements in chemically identical regions of the shell.⁸

The wide versatility of FTIR spectrometers in terms of the sample types that can be studied, from bones to cells to isolate molecules, has made FTIR spectrometers a very powerful and widely embraced tool in the biological sciences community that will only continue to develop with improvements in the analysis methods for large datasets and greater degrees of hardware automation.