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Chapter 5 : Analytical Imaging and Microscopy Techniques
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).
Images may be collected in the xy or xz plane, or a series of images may be collected in either the xy or xz plane. These photomicrographs show images collected in both the xy and xz planes in a biofilm community by using negative staining so that cells appear as dark objects (A) or positive staining so that cells appear as bright objects on a dark background (B). The location of the xz section is shown by the location of the horizontal line in each xy image in each panel.
A series of confocal micrographs taken at the same location in a sample of an algal mat. These xy images provide an illustration of multichannel imaging showing (A) an autofluorescence signal (algal chloroplasts), (B) reflection imaging of colloidal particles, and (C) an image of exopolymer labeled with the fluor-conjugated lectin concanavalin A-FITC. Reprinted from reference 141 with permission from the publisher.
(a) Analyses of CLSM confocal image stacks illustrating the application of basic image-processing steps to the analysis of a three-channel (green, red, and far red) fluorescence emission CLSM image stack of a river biofilm. The three stacks are used for the determination of algal (A, D, and G), bacterial (B, E, and H), and polymeric (C, F, and I) biomasses. The series shows the primary image (A, B, and C), application of segmentation to identify objects (D, E, and F), and application of dilation and erosion functions to eliminate noise in the images prior to determination of the number of object pixels in each category (G, H, and I). (b) Graphs showing sample data extracted from the image stacks as a bar graph of the percent biomass in each category and a pie chart of the proportional distribution of the measured parameters.
Illustration of the application of difference imagery to detect and quantify the algal and cyanobacterial signals and biomass in an LSM image series.
CLSM micrograph showing the three-color stereo pair created from the combination of images shown in Fig. 3 . The image shows the relative positions of algal and bacterial cells and their surrounding polymer matrix.
CLSM data set for protozoa grazing on a heterotrophic biofilm. The original data set is compared with the result obtained after blind deconvolution. (a) Maximum intensity projection (MIP) of original data; (b) MIP of blind deconvolution data; (c) 3-D isosurface reconstruction of original data; (d) 3-D isosurface reconstruction of deconvolved data; (e) zoomed region of interest from panel c; (f) zoomed region of interest from panel d. Color allocations are as follows: green, lectin-specific glycoconjugates stained with Aleuria aurantia lectin labeled with the Alexa-488 fluorochrome; red, bacterial cells stained with the nucleic acid-specific stain SYTO 60. Scale bars and grid boxes, 10 µm. Image data were recorded and deconvolution was calculated and projected by Christian Staudt.
(Top) Confocal micrographs illustrating the effects of selected pharmaceuticals on biofilm composition and architecture for control, carbamazepine, caffeine, furosemide, and ibuprofen after 8 weeks of development. The color wheel indicates the assignment of colors to bacteria (green), EPS and cyanobacteria (red), and other photosynthetic organisms (blue). (Bottom) Results of image analyses of confocal laser micrographs illustrating the effects of the pharmaceuticals on the proportional distribution of biomass of algae, cyanobacteria, and bacteria in the river biofilms. Reprinted from Lawrence et al. ( 132 ) with permission from the publisher.