Near-Field Scanning Optical Images of Bacteria
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Near-Field Scanning Optical Images of Bacteria Ana M. de Paula, Juliana A. Toledo, Haroldo B. Silva, and Gerald Weber ´ Laborat´orio de Nano-Espectroscopia Optica, Universidade S˜ao Francisco, Av. S˜ao Francisco de Assis 218, 12916-900 Braganc¸a Paulista-SP, Brazil. ABSTRACT Near field scanning optical microscopy (NSOM) and spectroscopy techniques were used to study the shape and the cell membrane details in bacteria. We present transmission and topographic images for the bacteria Pseudomonas aeruginosa using the Aurora NSOM from ThermoMicroscopes. The P. aeruginosa has been widely studied due to its clinical importance in many infectious diseases. The samples were stained by the Gram method and we measured the absorption of laser light at 488 nm by the dye (Safranin) fixed at the bacteria membrane. To obtain good images we had to improve the sample preparation in order to obtain isolated bacteria at the microscope slab. This was achieved using individual colonies, from a dry growth procedure, diluted in physiologic solution. Comparison of the topographic and transmission images give information on the shape and details of the absorption of the laser light by the cell membrane. The results show patterns that depend on the thickness and shape of the membrane, thus revealing details of the cell membrane with nanometer resolution. These results could be an important tool for instance in studies of the effects of antibiotics on the cell membrane. INTRODUCTION Near field scanning optical microscopy (NSOM or SNOM) offers the possibility of obtaining optical spectroscopy with nanometer resolution [1–4]. The resolution is obtained by coupling light through an optical fiber probe with a small aperture (typically 50 nm) that scans the sample at near field, i.e., at distances smaller than the light wavelength. The use of different optical spectral techniques such as fluorescence, Raman, absorption and reflection simultaneously with topographic images has opened up a variety of new applications in all material science areas. The chemical identification of different materials by optical spectroscopy at localized sample positions has been demonstrated to be an important tool in studying biological materials. Several cell biology applications have been reported [5–9]. In this paper we present near field scanning optical absorption and topographic images for the Gram-negative bacteria Pseudomonas aeruginosa. The P. aeruginosa has been widely studied due to its clinical importance in many infection diseases [10–12]. The bacteria were stained by the Gram method and we obtain the absorption image for the dye at the cell membrane. Comparison of the topographic and transmission images could give information on the shape and details of the absorption of the laser light by the cell membrane. The results show patterns that depend on the thickness and shape of the membrane, thus revealing details of the cell membrane with nanometer resolution. For comparison we present also scanning electron microscopy images of the same samples. The obtain
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