Atoms in molecules oscillate when irradiated by infrared light. The particular light frequencies that drive these vibrations are absorbed by molecules, and depend on the molecules’ chemical structure and environment. The infrared absorption spectrum of a sample can therefore be used as a molecular fingerprint by which to characterize its chemical composition. This has made infrared spectroscopy a widespread analytical technique. However, infrared spectra are difficult to measure for low concentrations of analytes and for samples in water. Writing in Nature, Pupeza et al.1 present a concept for infrared spectroscopy that promises to alleviate these limitations.

Read the paper: Field-resolved infrared spectroscopy of biological systems

Infrared light was discovered2 as a result of the problem it caused William Herschel while he was making astronomical observations of the Sun — it created a disturbing heating sensation in his eye that he wanted to filter out. Today, however, the benefits of infrared radiation for a multitude of analytical purposes are widely appreciated. Its applications range from the detection of molecules in outer space3,4, including that of water on Mars5, to deciphering the molecular mechanisms of proteins in living organisms6,7. In the everyday world, it is used in food analysis6,8 and in forensic police investigations6,9, for example. Much research is being done to bring infrared spectroscopy to the clinic, because the analysis of biological tissue and body fluids can be used to detect and diagnose disease6,7,10.

One of the main obstacles to the infrared analysis of biological samples is the strong absorption of infrared radiation by water — a problem that limits the sample thickness to less than 10 micrometres for most purposes. This issue also makes it difficult to add aqueous solutions of reagents (such as acids or salts) to samples to manipulate the state of molecules in the sample. Such manipulations are desirable, for example, for studying the binding of small molecules to proteins, and are standard practice when using ultraviolet or visible spectroscopy. Furthermore, because infrared radiation is absorbed by water, samples must often be concentrated or dried.

Snapshots of vibrating molecules

Pupeza and colleagues report a solution to this problem. They irradiate samples with an ultrashort pulse (on the scale of femtoseconds; 1 fs is 10–15 seconds) of mid-infrared light. Specific frequencies of the light are absorbed by sample molecules, generating vibrations. These vibrations continue after the pulse has ended, and last until the vibrational energy is dissipated to the environment (which takes a few picoseconds; 1 ps is 10–12 s). Because the vibrating atoms carry partial electrical charges, their oscillations generate electromagnetic radiation, similar to the way in which oscillating electrons produce electromagnetic radiation in an antenna. The generated radiation has the same frequency as that of the molecular vibrations, and so carries information about all of the sample molecules — the authors therefore call it a global molecular fingerprint. It is measured using a second ultrashort pulse of light, this time in the near-infrared spectral range, through a method called electro-optic sampling11.

The authors’ approach is conceptually different from conventional absorption measurements. In absorption spectroscopy, the signal is sensed only indirectly, from the light that does not interact with the sample (Fig. 1a). Weak absorption is therefore very difficult to detect, because it changes the intensity of the transmitted light only marginally. Theoretically, the detection of weak absorbers could be improved by increasing the intensity of the incident light, but commonly used infrared detectors become less sensitive at higher light intensities12, imposing a practical limit on the maximum light intensity that can be used. By contrast, Pupeza et al. detect the signal of interest — the radiation emitted from the vibrating molecules — directly (Fig. 1b). This is analogous to the difference between absorbance and fluorescence measurements in the visible spectral range: fluorescence measurements are the more sensitive because they detect a signal directly from the sample, and can even detect it from a single molecule.

Figure 1 | A fresh approach for obtaining infrared spectra. a, In conventional infrared spectroscopy, molecules are irradiated with infrared light. They absorb certain frequencies of the light, which causes them to vibrate. The signals of interest are the absorption ‘troughs’ in the transmitted light spectrum, but these change the overall intensity of the transmitted light only marginally when the samples are highly diluted, limiting the sensitivity of this technique. b, Pupeza et al.1 irradiate analytical samples with ultrashort bursts of infrared light, again causing molecules in the sample to vibrate. These vibrations continue after the pulse has ended, and generate infrared radiation, shown here as a ‘tail’ that trails after the pulse. This tail is analysed to determine the infrared spectrum of the molecules. Because the experimental signal is emitted light and is detected directly, this method can be more sensitive than absorption infrared spectroscopy.

Pupeza and colleagues demonstrate the high sensitivity of their approach in various ways. For example, they were able to detect 40-fold lower concentrations of a compound in solution, and to better distinguish between two similar compounds, than when using absorption spectroscopy. They also obtained spectra of biological samples that block nearly all of the incoming light (in one case, at least 99.999%). Thus, the new approach senses light where currently used methods see only darkness. This is an impressive achievement, and might alleviate both of the main problems of conventional infrared spectroscopy: sensitivity and strong infrared absorption by water. It will simplify sample preparation in many cases by removing the need for sample concentration or drying, and will open up new applications — particularly those involving aqueous biological samples.

The authors suggest several ideas for taking the method further, such as by increasing the power of the laser used to irradiate the sample. It is to be hoped that such measures will further narrow the technological gap that at present prevents the method from achieving the ultimate goal of single-molecule sensitivity in bulk water. Other challenges will be to increase the spectral range of the measurements to include the shorter wavelengths at which prominent and diagnostically useful signals are found for proteins, lipids and nucleotides, and to develop a spectrometer suitable for commercialization at a competitive price.