Doktoratskolleg MEIBio - Molecular and Elemental Imaging in Bioscience
> Zum Inhalt

PhD Fellow #4

Photoexpansion Microscopy: Combining Pulsed Mid-IR Laser Spectroscopy With Atomic Force Microscopy

Faculty: Bernhard Lendl

Mid-infrared (mid-IR) spectroscopy is a non-destructive and label free analytical technique for the analysis of bio-molecules and biological systems. Commercially available IR microscopes consisting of an optical microscope and an infrared spectrometer allow spatially resolved infrared spectroscopy. Such instruments are for example employed by pathologists to discern types of tissues in sections without the need for tedious staining. A general limitation of the spatial resolution achievable in optical techniques is given by the Rayleigh limit; for mid-IR wavelengths this limit is in the single digit micrometer range (1). The Rayleigh limit can be circumvented by using near-field instead of far-field detection of the infrared radiation. A current technique for near-field detection at mid-IR wavelengths is the AFMIR technique (2, 3). AFMIR, a setup newly applied for multimodal imaging, uses infrared lasers as light sources and a standard atomic force microscope (AFM) as a near-field detector. Short laser pulses are directed onto the sample and lead to a warming of the sample in regions of infrared absorption. This warming leads to local thermal expansion of the sample which can sensitively be detected by the AFM (4-6).

External-Cavity Quantum Cascade Laser (EC-QCL) sources exhibit a broad spectral tuning range and a high repetition rate. The high repetition rate allows to modulate the laser at the resonance frequency of the cantilever and thereby to reduce the thermal stress of the sample. By exploiting plasmon resonance effects even films of 10 nm thickness can be measured (7) (Figure).

During the proposed PhD studies an AFMIR setup will be realized.

 

  • This instrument will allow to perform high resolution spectroscopy in several wavelength ranges containing biologically relevant information (e.g. carbohydrate range, amide I range).
  • Additionally this setup can be used to analyze the influences of sample geometry and material properties on the AFMIR signal by using lithographically fabricated samples.
  • Since AFMIR spectroscopy allows the analysis of 10 nm thick layers it can be used to analyze biological membranes.

While high resolution AFM analysis of membrane topologies is well established the AFMIR technique will be able to add optic information regarding membrane constituents (lipids, proteins, carbohydrates,...) directing this approach towards new instrumentation for multimodal imaging.

1. F. Lu and M. Belkin 2011, Mid-infrared absorption microscopy with λ / 100 spatial resolution using tunable quantum cascade lasers in OSA/CLEO, pp. 4-5.
2. A. Dazzi, 2004. Theoretical study of an absorbing sample in infrared near-field spectromicroscopy. Optics Comm, 235( 4-6):351-360.
3. A. Dazzi, C. B. Prater, Q. Hu, D. B. Chase, J. F. Rabolt, and C. Marcott. 2012. AFM-IR: Combining Atomic Force Microscopy and Infrared Spectroscopy for Nanoscale Chemical Characterization. Appl Spectrosc 66(12): 1366-1384.
4. C. Mayet, A. Dazzi, R. Prazeres, J.-M. Ortega, and D. Jaillard (2010). In situ identification and imaging of bacterial polymer nanogranules by infrared nanospectroscopy.," The Analyst 135(10): 2540-2545.
5. C. Policar, J. B. Waern, M.-A. Plamont, S. Clède, C. Mayet, R. Prazeres, J.-M. Ortega, A. Vessières, and A. Dazzi. 2011. Subcellular IR imaging of a metal-carbonyl moiety using photothermally induced resonance. Angew Chemie (International ed. in English) 50(4): 860-4.
6. A. Dazzi, R. Prazeres, F. Glotin, J. M. Ortega, M. Al-Sawaftah, and M. de Frutos 2008. Chemical mapping of the distribution of viruses into infected bacteria with a photothermal method. Ultramicroscopy 108(7): 635-41.
7. F. Lu and M. Belkin (Oct 2012) Plasmonic-enhanced infrared photoexpansion nano-spectroscopy using tunable quantum cascade lasers," no. i, p. 84660I-84660I-6.

Collaboration:

 

  • In the area of AFM infrared spectroscopy the Lendl group is in research co-operation with Prof. Belkin at University of Texas at Austin (Austin, TX) as well as with Dr. Andrea Centrone at NIST (Gaithersburg, MD).
  • In order to assess the imaging quality achieved on biological samples it will be necessary to compare the obtained data in reference to other established techniques such as fluorescence (G. Schütz, TU Vienna).
  • Complimentary to AFMIR, Raman spectroscopy can be coupled to ultramicroscopy and mass spectrometry. For this purpose it is planned to join forces with the research activities of G. Schütz, H.U. Dodt as well as M. Marchetti-Deschmann (all TU Vienna) as these research groups have established track records focussing on biological samples.