Chapter 5 : Analytical Imaging and Microscopy Techniques

Abstract:

Examination of microorganisms in their natural habitat may be achieved most effectively through the application of a variety of microscope-based techniques. A major goal of the use of microscopic techniques is to achieve minimum disturbance of the system under observation. The major guidelines for all types of laser scanning microscopy (LSM) imaging are to obtain an image using the lowest-intensity laser and the smallest-pinhole aperture to minimize photodamage, optimize image quality (i.e., signal-to-noise ratio), and minimize the thickness of the optical section. Probes useful for fluorescence microscopy may be divided into three different types. Intrinsic probes are already present inside the sample (e.g., pigments such as chlorophyll or phycoerythrins and phycocyanins). Extrinsic probes are those which bind directly to a target (e.g., the general nucleic acid stains, such as 4 ,6 -diamidino-2-phenylindole [DAPI] or the SYTO series). Extrinsic covalently bound probes are usually high-molecular-weight molecules with a high specificity but no fluorescence (e.g., antibodies, lectins, and gene probes). Fluorescence in situ hybridization (FISH) of oligonucleotide probes to specific bacteria has previously been used in conjunction with confocal laser scanning microscopy (CLSM) and epifluorescence microscopy to document microbial diversity in a range of environments, including sewage sludge, river biofilms, and the rhizosphere. A number of emerging microscopy techniques, particularly in fluorescence imaging, may be applied to microbiological samples. These include FLIM, FCS, coherent anti-Stokes Raman scattering (CARS) microscopy, second harmonic imaging microscopy, 4Pi microscopy, stimulated emission depletion microscopy, and near-field scanning optical microscopy (NSOM).