Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths ( l) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 mum (approximate tolambda1/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.

Very high resolution near-field chemical imaging using an infrared free electron laser

Giorgio Margaritondo

High resolution infrared imaging of thin films and biological systems is one of the most challenging experimental problems in contemporary science and technology. In this work, we have for the first time successfully tested a novel high resolution approach, based on a spectroscopic version of scanning near-field optical microscopy (SNOM). The coupling of the Vanderbilt Free Electron Laser tunable infrared radiation to SNOM apparatus enabled us to clearly reveal different chemical constituents on a growth medium for biofilm. The images were obtained by SNOM detection of reflected 6.95 mum photons, corresponding to the stretch absorption of sulfur and nitrogen compounds, constituents of the growth medium. We attained a lateral resolution of 0.2 mum (lambda/35), well below the diffraction limit of classical microscopy.

2002

Spectroscopic scanning near-field optical microscopy with a free electron laser: CH2 bond imaging in diamond films

Giorgio Margaritondo

Hydrogen chemistry in thin films and biological systems is one of the most difficult experimental problems in today's science and technology We successfully tested a novel solution, based on the spectroscopic version of scanning near-field optical microscopy (SNOM). The tunable infrared radiation of the Vanderbilt free electron laser enabled us to reveal clearly hydrogen-decorated grain boundaries on nominally hydrogen-free diamond films. The images were obtained by SNOM detection of reflected 3.5 mum photons, corresponding to the C-H stretch absorption, and reached a lateral resolution of 0.2 mum, well below the lambda /2 (lambda = wavelength) limit of classical microscopy.

2001

Scanning near-field infrared microscopy and spectromicroscopy applied to nano-systems and cells

Dusan Vobornik

This thesis further explores the possibilities of scanning near-field optical microscopy (SNOM) in both materials and life sciences. Two experimental SNOM setups were developed: one designed for infrared spectroscopy applications and the other for the imaging of fluorescently labeled samples. The results of the experiments that were conducted with both setups are presented and analyzed in this thesis. Diffraction limits the resolution of lens-based microscopes to a value close to that of the wavelength of the light that is used to illuminate the samples. SNOM instruments overcome this limit by probing the near-field light — the light that remains within close vicinity of the sample and decays exponentially away from it. The SNOM concept and instrumental design are described. A comparison of SNOM with other novel microscopies (electron, atomic force, and scanning tunneling microscopies) outlines the main advantages of SNOM — the sole optical microscopy technique with a nanometer resolution. Aperture-based infrared SNOM (IR-SNOM), performed in the spectroscopic mode using the Vanderbilt University free electron laser, recently started delivering spatially resolved information on the distribution of chemical species and on other laterally fluctuating properties. The IR-SNOM combines the IR spectroscopy's chemical specificity and the SNOM's high optical and topographical resolutions. The IR-SNOM experimental setup is described in detail and results from the study of cells, boron nitride and lithium fluoride thin films are presented and analyzed. They demonstrate the great potential of this new technique. An overview of the development of IR-SNOM, as well as a consideration of its future possibilities are presented. The newly built SNOM instrument at EPFL was used for experiments that are complementary to the aforementioned IR-SNOM experiments. Specifically, the new SNOM enabled the study of fluorescently marked samples. Images of fluorescently labeled cells and carbon nanotubes are presented and analyzed. The results further demonstrate the exciting possibilities of SNOM.

EPFL2005

Scanning near-field infrared microscopy and spectromicroscopy applied to nano-systems and cells

Dusan Vobornik

EPFL2005