Infrared Sub-Cellular Imaging with an Atomic Force Microscope

The existing tools available to perform infrared spectroscopy and microscopy at the nanometer scale are limited considering all the different near-field microscopes. However AFM-IR, a new infrared spectromicroscope coupling an Atomic Force Microscope (AFM) to a tunable laser, allows researchers to derive chemical information on a scale not previously possible. The assignation of bands of absorption is non-ambiguous allowing spectroscopists to use AFM-IR spectra as easily as those obtained using classical infrared methods.

The principle of AFM-IR¹ is to couple the AFM in contact mode with a pulsed tunable laser (Figure 1). A sample is placed on an infrared-transparent prism and then irradiated with the laser. When the laser wavelength is tuned on the absorption bands of the sample, the absorbed laser light causes a photothermal temperature rise in the absorbing regions of the sample. As the temperature increases due to IR absorption, the sample expands. The local thermal expansion is monitored with the tip of the AFM. The rapid thermal expansion of the sample generates a force impulse that drives the cantilever into oscillation. So each time the light pulse is absorbed and heats the sample, the cantilever oscillates at its resonance frequency. The amplitude is directly proportional to the energy absorbed², thus leading to absorption spectra that are readily correlated to bulk IR spectroscopy techniques like FTIR. Compared to FTIR, the sensitivity of the AFM-IR technique is able to chemically identify samples on the size scale of tens of nanometer.

Figure 1. Schematic of AFMIR technique

The AFM-IR technique has been used in our research center (Laboratoire de Chimie Physique, Orsay, France) for five years. The experiments were set up and run at the Centre Laser Infrarouge d’Orsay and now provides a permanent beamline. CLIO is run in a rather unusual way: It allows us to offer our systems to outside users from other research groups. The specification of the source is to be a free electron laser tunable from 3 to 150 µm. Access is managed by a program committee similar to those at synchrotron centers. It is in this context that we have been able to collaborate on several projects in different areas, particularly in biology^3,4,5,6,7, and in nanophotonics.^8,9,10

Application example in microbiology : PHB location into Rhodobacter capsulatus⁶

Rhodobacter capsulatus is a purple nonsulfur photosynthetic bacterium, which produces a polymer, polyhydroxybutyrate (PHB), for its energy storage under vesicle inclusion shape. PHB belongs to a class of polyesters and has been used for several years in the production of plastics having similar mechanical and thermoplastic properties to those of polyethylene and polypropylene but with the advantage of being able to use renewable resources. The presence of PHB can be probed in the mid-infrared domain by the presence of specific absorption bands, in particular around 1740 cm^-1 (C=Os of ester), easily distinguishable from other bacteria bands : Amide I at 1660 cm^-1, Amide II at 1550 cm^-1.

The top images in Figure 2 display the topography of bacteria obtained by classic AFM. The bottom images show the corresponding chemical cartography of PHB (at 1740 cm^-1). On all maps, we have localized round areas where the signal is more intense (red domains). These domains correspond to PHB granules inside bacteria (Figure 2 d, e, f). On each chemical map, we can estimate the size of granules by estimating the width at the half height. Figure 2d reveals a big round granule of 210 nm diameter and long shape one of only 50 nm large (top of the image). This long shape is very likely the result of small spherical PHB vesicles lined up close to the membrane. Figure 2e shows a bacterium without PHB vesicle. This suggests that under these growing conditions, all Rhodobacter capsulatus bacteria do not necessarily produce PHB. Figure 2f (zoom of Figure 2e) reveals that the absorbing area is in fact composed by two adjacent vesicles of different sizes.

Figure 2a. AFM topography of one single Rhodobacter capsulatus.

Figure 2b. AFM topography of two seperated bacteria Rhodobacter.

Figure 2c. AFM zoom on the lowest bacterium localized on b).

Figure 2d. Chemical mapping of PHB (at 1740 cm^-1) of the corresponding topogaphy a).

Figure 2e. Chemical mapping of PHB of b).

Figure 2f. Chemical mapping of PHB of c).

We have studied the spectroscopy response of the vesicle (Figure 2f) and compared it with the FTIR spectrum of the bacteria culture (Figure 3). The spectrum on a single bacterium (in red Figure 3a) was measured by positioning the tip of the AFM directly on the maximum of the signal using the chemical mapping of PHB (as pointed by Figure 3b). We observe an intense band of C=O of ester (centered at 1740 cm^-1) whereas the Amide I band at 1660 cm^-1 appears weaker and noisy, demonstrating the PHB nature of the hot spot mapping from Figure 3b. The second spectrum was recorded by positioning the tip at point B (Figure 3) at the border of the absorption vesicle. The spectrum shows a better signal for the Amide I that is similar to the FTIR spectrum of the bacteria culture (Figure 3 in green). The intensity of the PHB in that case has decreased compared to the previous position, which is consistent with the PHB chemical mapping. When the tip is positioned to C (Figure 3), out of the vesicle, the AFM-IR spectrum does not show the C=O band (in violet Figure 3).

Figure 3a. Comparison between local (A in red, B in orange, C in violet) AFM-IR spectra and FTIR spectrum (in green) of the bacteria culture.

Figure 3b. Bacterium chemical mapping of C=O of ester band with corresponding position (A, B, C) of the spectra measurements.

These results are of the utmost interest as AFM-IR is a non-invasive technique that can be applied directly to the study of single cells. Thanks to this technique, nano-resolution is now attainable for imaging using IR radiation. This makes IR imaging possible at the subcellular scale, a breakthrough in IR-mapping. Spectromicroscopy represents a powerful tool to determine ultra-local composition in cellulo

Released in 2010, AFM-IR is now available as a commercial benchtop instrument: the nanoIR, developed and sold by Anasys Instruments Inc.

References and Further Reading

1. A. Dazzi, R. Prazeres, F. Glotin, J.M. Ortega, Opt. Lett. 30, 2388 (2005).
2. A. Dazzi, F. Glotin, and R. Carminati, J. Appl. Phys. 107, 124519 (2010)
3. A.Dazzi, R.Prazeres, F.Glotin, J.M.Ortega, Infrared Physics and Technology, 49, 113 (2006).
4. A.Dazzi, R.Prazeres, F.Glotin, J.M.Ortega, M.Alsawaftah, M.De Frutos, Ultramicroscopy 108, 635-641, (2008).
5. C. Mayet, A. Dazzi, R. Prazeres, F. Allot, F. Glotin, J.M. Ortega, Opt. Lett. 33,1611-1613 (2008).
6. C. Mayet, A. Dazzi, R. Prazeres, J.-M. Ortega , D. Jaillard, Analyst 135, 2540-2545 (2010).
7. C. Policar, J. B. Waern, M. A. Plamont, S. Clède, C. Mayet, R. Prazeres, J.-M. Ortega, A. Vessières, and A. Dazzi, Angewandte Chemie International Edition,Vol 50, Issue 4, 860–864, (2011).
8. J.Houel, S.Sauvage, P.Boucaud, A.Dazzi, R.Prazeres, F.Glotin, J.M.Ortéga, A.Miard, A.Lemaître, Phys Rev Lett 99, 217404 (2007).
9. J. Houel, E. Homeyer, S. Sauvage, P. Boucaud, A. Dazzi, R. Prazeres, J.M.Ortega, Optics Exp., 17, 10887-10894 (2009).
10. S. Sauvage, A. Driss, F. Réveret, P. Boucaud, A. Dazzi, R. Prazeres, F. Glotin, J.-M. Ortéga, A. Miard, Y. Halioua, F. Raineri, I. Sagnes and A. Lemaître , Phys. Rev. B 83, 035302 (2011).