Infrared Nano-spectroscopy Intracellular Measurements: the protein distribution in HeLa Cell

The infrared spectroscopy is an experimental technique which allows to characterize materials thanks to their bio-chemistry composition: focusing the infrared radiation on the sample we can excite the vibrational mode of a molecule and, in this way, we can obtain a sort of fingerprint of the molecule.

If we couple the infrared spectroscopy to imaging techniques, we can obtain a powerful instrument which allows us to localize the position of the different chemical components. However, in cases like cellular imaging, we need a high spatial resolution which can’t be obtained with conventional microscopy techniques, due to the diffraction limit.

In order to solve this problem we can use a near-field technique which allows us to go beyond the diffraction limit and to obtain infrared images with nanometric resolution: in fact, in biological samples is very difficult to separate the information about the topography and dishomogeneity from the information of interest, i.e. the infrared absorption.

In my talk I will show a new near-field technique which couples two well known experimental techniques: the infrared spectroscopy and the atomic force microscopy.

In fact, by coupling an AFM to a quantum cascade laser which emits in the mid-infrared, we can make use of the near-field photo-thermal effect (PTIR): the infrared radiation excites the vibrational modes of molecules and inthis way it heats the sample locally, hence inducing a thermal expansion. This expansion can be recorded by the AFM.

This new technique has a lot of advantages in biological field because it has a spatial resolution higher than conventional microscopy techniques (~100nm) and in this way we don’t risk to damage the sample. For these reason we can use the PTIR technique to localize the proteins inside cells.

The HeLa Cells were be treated so that they can express their proteins and form some agglomerate called Stress Granules. Stress Granules were measured with PTIR spectroscopy and analyzed by our group developing the experimental protocol for data analysis.