We present a method for obtaining a position-dependent absorption coefficient from near-field scanning optical transmission microscopy. We show that the optical transmission intensity can be combined with the topography, resulting into an absorption coefficient that simplifies the analysis of different materials within a sample. The method is tested with the dye rhodamine 6G, and we show some analysis in biological samples such as bacteria KIebsiella pneumoniae and Pseudomonas aeruginosa. The calculated absorption coefficient images show important details of the bacteria, in particular for P. aeruginosa, in which membrane vesicles are clearly seen.

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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.