10 ft m

FIGURE 6.1 (A) Image shows a silicone nitride window attached to a rotating annular biofilm reactor, and detail in inset shows window and central x-ray transparent region for STXM imaging; (B) CLSM image of x-ray transparent region showing biofilm development on the window.

systems suitable for application in the study of flocs and biofilms. Specific techniques include those for detection and quantification of cellular and polymeric compounds in biofilms.9'16'27 In addition, Neu et al.28 demonstrated that 2P-LSM could be effectively applied to the study of highly hydrated microbial systems such as flocs and that a range of fluorescent reporters for both cell and exopolymer identity could be applied in combination with this imaging approach. Figure 6.2 provides a comparison of the excitation for 1P versus 2P for the common fluor fluorescein illustrating the different response of the fluor in the two forms of LSM. Extensive details of these microscopy techniques and their use in combination with biofilms and flocs are provided in Lawrence et al.27 Neu,1 Lawrence and Neu,29 and Lawrence et al.30 CLSM Limitations

A limitation of 1-photon excitation is laser penetration of samples (excitation) and detection of emission signal in thick samples. This problem is very much dependent upon the density and light scattering properties of the sample. Consequently thick samples have to be embedded and physically cut into slices using embedding resins or cryosectioning. 2P-LSM Limitations

The major problems are the overall stability of the laser system, maintenance of signal intensity, and excessive noise in the image. In addition, images may be degraded by reaction of the light source with the substratum or mounting materials causing, for example, streaks in the image due to adsorption of infrared light (e.g., plastics). Although laser penetration is better (twofold) in 2P-LSM over CLSM, light scattering in thick biological samples remains a problem.

Wavelength (nm)

FIGURE 6.2 Comparison of the 1P and 2P emission for fluorescein when excited at wavelengths between 400 and 900 nm.

Wavelength (nm)

FIGURE 6.2 Comparison of the 1P and 2P emission for fluorescein when excited at wavelengths between 400 and 900 nm.

6.2.4 Synchrotron Radiation (Soft x-ray Imaging)

Scanning transmission x-ray microscopy (STXM) is a powerful tool that may be applied to fully hydrated biological materials. This is due to the capacity of soft x-rays to penetrate water and have minimal radiation damage relative to electron techniques. In addition, soft x-rays interact with nearly all elements and also allow mapping of chemical species based on bonding structure.31 Soft x-ray microscopy also provides suitable spatial resolution and chemical information at a microscale relevant to bacteria. Most importantly, the method uses the intrinsic x-ray absorption properties of the sample eliminating the need for the addition of reflective, absorptive, or fluorescent probes and markers which may introduce artifacts or complicate interpretation. Figure 6.3 shows the representative absorption spectra for protein, nucleic acid, saccharide, lipid, and calcium carbonate. The potential of soft x-rays for imaging early stage Pseudomonas putida biofilms using a full field transmission x-ray microscope with synchrotron radiation was demonstrated by Gilbert et al.32 They measured at single photon energy and did not explore the analytical capability of x-ray microscopy. Lawrence et al.25 demonstrated the application of analytical soft x-ray microscopy to map protein, nucleic acids, lipids, and polysaccharides in biofilm systems. Hard

FIGURE 6.3 C 1s NEXAFS spectra of protein (albumin), polysaccharides (sodium alginate), lipid(1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine), and nucleic acid (calf thymus DNA). All spectra except that of DNA were recorded with the ALS 7.0.1 STXM. The spectrum of DNA was recorded on ALS 5.3.2 STXM. (Copyright American Society for Microbiology, Lawrence, J.R. et al. Appl. Environ Microbiol: 69: 5543-5554, 2003.)


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