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Chapter 3 : Laser Scanning Microscopy

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Laser Scanning Microscopy, Page 1 of 2

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Abstract:

This chapter provides a basis for understanding laser scanning microscopy (LSM) and the approaches that may be used to apply high-resolution digital microscopy to the study of bacteria. Additional reviews of image analysis, digital imaging, and LSM for microbiology applications are also provided in this chapter. The major focus of most courses is cell biology, although the information is of use and techniques are broadly transferable across disciplines. In practice, one of the appealing aspects of fluorescence microscopy is the lack of a requirement for fixation, dehydration, and the air drying of samples required by some other techniques. One of the goals of this approach is to examine living bacterial cells, aggregates, and biofilms. Changes in any of the microscope settings will alter the section thickness, the brightness, and the apparent size of the objects being imaged. The major advantage of LSM is the capacity to collect a series of images that allow the user to obtain 3-D spatial information. The chapter gives general protocol and considerations of lectin staining. A number of approaches have been applied to study diffusion in bacterial biofilms and serve to illustrate the application of kinetic analyses using LSM systems. The major limitation of all light microscopic systems and particularly LSM is poor axial resolution. In LSM there are both 2-D- and 3-D-related considerations regarding sampling. Application of LSM techniques results in the creation of vast image and data sets.

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3

Key Concept Ranking

Environmental Microbiology
0.6615973
Microbial Ecology
0.6596852
Confocal Laser Scanning Microscopy
0.6006832
Fluorescence Lifetime Imaging Microscopy
0.5353967
Microbiological Methods
0.48181006
0.6615973
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Figures

Image of FIGURE 1X
FIGURE 1X

1P and 2P LSM images of a fluidized bed reactor biofilm which degrades EDTA and is growing on pumice. The specimen was stained with SYBR green, and serial sections were taken by using both LSM systems on the same sample sequentially. Images show enhanced resolution of the 2P system relative to that of the 1P system with a 39-μm-thick biofilm specimen.

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 2
FIGURE 2

Graphs showing the nature of excitation-emission spectra for an ideal series of green, red, and far-red-emitting fluors (A) and problematic fluors where excitation (Ex) and emission (Em) peaks overlap, resulting in potential cross talk or bleed through of the signal into adjacent imaging windows (B).

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 3
FIGURE 3

1P LSM images showing a direct single-scan image (A) and the influence of image averaging on the quality and signal- to-noise ratio of an LSM image (B).

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 4
FIGURE 4

1P LSM micrographs showing the nature of the 3-D stereo pair (A) and a single (B) and a single (C) scan through a microbial biofilm stained with the nucleic acid stain SYTO9 (Molecular Probes).

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 5
FIGURE 5

Series of 1P LSM images showing the monitoring of pH by using a dually labeled (pH-sensitive fluorescein and pHinsensitive rhodamine) 10,000-molecular-weight dextran in a microbial biofilm. (A) pH-sensitive imaging of fluorescein. (B) pHinsensitive fluorescence of the rhodamine. (C) Grayscale representation of the standard curve for pH versus the ratio of images A and B. (D) Contour map showing the distribution of pH levels within the microbial biofilm.

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 6
FIGURE 6

Gallery presentation of a Z series taken through a microbial biofilm stained with SYTO9.

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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Image of FIGURE 7
FIGURE 7

3-D stereo pair presentation of the Z series shown in Fig. 6 .

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
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References

/content/book/10.1128/9781555817497.chap3
1. Amann, R.,, R. Snaidr,, M. Wagner,, W. Ludwig,, and K. H. Schleifer. 1996. In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178:34963500.A useful example of application of multiple fluors.
2. Amann, R. I.,, W. Ludwig,, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143169. Excellent review of both FISH and CLSM approaches with good color stereo images of microbial specimens.
3. Axelrod, A.,, D. E. Koppel,, J. Schlessinger,, E. Elsen,, and W. W. Webb. 1976. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16:10551069.A more technical paper which provides the basis for using FRAP to calculate the mobility of molecules.
4. Birmingham, J. J.,, N. P. Hughes,, and R. Treloar. 1995. Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods. Phil. Trans. R. Soc. Lond. B 350:325343.A practical application of FRAP in microbial samples.
5. Blackburn, N.,, A. Hagstrom,, J. Wikner,, R. Cuadros- Hansson,, and P. K. Bjornsen. 1998. Rapid determination of bacterial abundance, biovolume, morphology, and growth by neural network-based image analysis. Appl. Environ. Microbiol. 64:32463255.An article for those with a greater interest in more sophisticated handling of images and data extraction.
6. Blonk, J. C. G.,, A. Don,, H. van Aalst,, and J. J. Birmingham. 1993. Fluorescence photobleaching recovery in the confocal scanning laser microscope. J. Microsc. 169:363374. Technical paper providing more complex mathematical treatment of the data obtained through imaging FRAP.
7. Boleti, H.,, D. M. Ojcius,, and A. Dautry-Varsat. 2000. Fluorescent labelling of intracellular bacteria in living host cells. J. Microbiol. Methods 40:265274.Technical information on the use of CellTracker with bacteria.
8. Bright, G. R.,, and D. L. Taylor,. 1986. Imaging at low light level in fluorescence microscopy, p. 257288. In D. L. Taylor,, A. S. Waggoner,, F. Lanni,, and R. Birge (ed.), Applications of Fluorescence in the Biomedical Sciences. Liss Inc., New York, N.Y. An older book that still provides an excellent overview of and entry point into the very large cell biology literature which contains a vast array of vital information on fluors and techniques with potential for application in prokaryotic studies.
9. Bryers, J. D.,, and F. Drummond. 1998. Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photo-bleaching (FRAP). Biotechnol. Bioeng. 60:462473. Useful technical note on the FRAP method applied to microorganisms.
10. Caldwell, D. E.,, D. R. Korber,, and J. R. Lawrence. 1992. Confocal laser microscopy and digital image analysis in microbial ecology. Adv. Microb. Ecol. 12:167.The first comprehensive overview of applications and potential of 1P LSM and digital imaging focused on the area of microbial ecology; still provides a good starting point.
11. Caldwell, D. E.,, D. R. Korber,, and J. R. Lawrence. 1992. Imaging of bacterial cells by fluorescence exclusion using scanning confocal laser microscopy. J. Microbiol. Methods 15:249261.Technical note describing details of the fluorescent negative staining technique for bacteria and biofilms.
12. Caldwell, D. E.,, G. M. Wolfaardt,, D. R. Korber,, S. Karthikeyan,, J. R. Lawrence,, and D. K. Brannan,. 2001. Cultivation of microbial consortia and communities, p. 92100. In C. J. Hurst,, R. L. Crawford,, G. R. Knudsen,, M. J. McInerney,, and L. D. Statzenbach (ed.), Manual of Environmental Microbiology, 2nd ed. ASM Press, Washington, D.C.. Overview; features many devices for cultivation of bacteria and biofilms that are compatible with microscopic studies.
13. Choi, J.-W.,, B. F. Sherr,, and E. B. Sherr. 1999. Dead or alive? A large fraction of ETS-inactive marine bacterio-plankton cells, as assessed by reduction of CTC, can become ETS-active with incubation and substrate addition. Aquat. Microb. Ecol. 18:105115. A good example of validation of a fluorescence technique for natural bacterial populations and important considerations in the process.
14. Christensen, B. B.,, C. Sternberg,, J. B. Andersen,, L. Eberl,, S. Møller,, M. Givskov,, and S. Molin. 1999. Molecular tools for study of biofilm physiology. Methods Enzymol. 310:2042. Excellent overview of fluorescence-based techniques including those compatible with LSM.
15. Conn, P. M. 1999. Methods in Enzymology, vol. 307. Confocal Microscopy. Academic Press, New York, N.Y. A comprehensive compendium of papers on various areas of confocal microscopy with technical details and applications.
16. Coote, P. J.,, C. M.-P. Billon,, S. Pennel,, P. J. McClure,, D. P. Ferdinando,, and M. B. Cole. 1995. The use of confocal scanning laser microscopy (CSLM) to study the germination of individual spores of Bacillus cereus. J. Microbiol. Methods 21:193208. A good example of technical application of 1P LSM and fluorescent labeling.
17. Cullander, C. 1994. Imaging in the far-red with electronic light microscopy: requirements and limitations. J. Microsc. 176:281286.
18. De Beer, D.,, P. Stoodley,, and Z. Lewandowski. 1997. Measurements of local diffusion coefficients in biofilms by microinjections and confocal microscopy. Biotechnol. Bioeng. 53:151158. Excellent paper for those interested in calculation of diffusion from time course images of microinjected fluor-conjugated probes.
19. Decho, A. W.,, and T. Kawaguchi. 1999. Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin. BioTechniques 27:12461252. Useful starting point for combined use of hydrophilic resin embedding and LSM.
20. Florijn, R. J.,, J. Slats,, H. J. Tanke,, and A. K. Raap. 1995 Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177182.
21. Haugland, R. P. 1998. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes Inc., Eugene, OR. Comprehensive guide to the fluorescent products provided by Molecular Probes; includes much useful background information, excitation-emission spectra, and literature.
22. Hausner, M.,, and S. Wuertz. 1999. High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis. Appl. Environ. Microbiol. 65:37103713. A novel application of 1P LSM for in situ analyses of single-cell molecular events.
23. Heydorn, A.,, A. T. Nielsen,, M. Hentzer,, C. Sternberg,, M. Givskov,, B. K. Ersböll,, and S. Molin. 2000. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:23952407. Paper that provides a good entry point into the use of statistical considerations in imaging of microbial biofilms.
23.a. Hunter, R. C.,, and T. J. Beveridge. 2005. Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 71:25012510. A good example of ratiometric applications of fluorescent pH reporters and useful controls for these studies.
24. Kepner, R. L.,, and J. R. Pratt. 1994. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microb. Rev. 58:603615. Excellent overview of the literature to 1994 on fluorochromes, including applications and problems.
25. Korber, D. R.,, J. R. Lawrence,, M. J. Hendry,, and D. E. Caldwell. 1993. Analysis of spatial variability within mot+ and mot Pseudomonas fluorescens biofilms using representative elements. Biofouling 7:339358. Paper describes application of geostatistical methods to biofilm research and establishment of sampling requirements; also provides images of montages of large biofilm areas.
26. Laca, A.,, L. A. Garcia,, F. Argüeso,, and M. Diaz. 1999. Protein diffusion in alginate beads monitored by confocal microscopy. The application of wavelets for data reconstruction and analysis. J. Ind. Microbiol. Biotechnol. 23: 155165.
27. Lawrence, J. R.,, and T. R. Neu. 1999. Confocal laser scanning microscopy for analysis of microbial biofilms. Methods Enzymol. 310:131144. A brief overview of how to do 1P LSM with microbial biofilms and flocs; also examines specimen handling and upright versus inverted microscopes.
28. Lawrence, J. R.,, T. R. Neu,, and G. D. W. Swerhone. 1998. Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms. J. Microbiol. Methods 32:253261. Useful techniques paper describing how to use autofluorescence, nucleic acid staining, and lectin staining with digital imaging to quantify major biofilm components.
29. Lawrence, J. R.,, G. D. W. Swerhone,, and Y. T. J. Kwong. 1998. Natural attenuation of aqueous metal contamination by an algal mat. Can. J. Microbiol. 44:825–832. The paper provides a useful example of combining reflectance, fluorescence, and autofluorescence techniques.
30. Lawrence, J. R.,, G. M. Wolfaardt,, and T. R. Neu,. 1998. The study of biofilms using confocal laser scanning microscopy, p. 431465. In M. H. F. Wilkinson, and F. Schut (ed.), Modern Microbiological Methods Series, vol. •. Digital Analysis of Microbes. Imaging, Morphometry, Fluorometry and Motility Techniques and Applications. John Wiley and Sons, Sussex, United Kingdom. A comprehensive overview of applications of 1P LSM to microbial biofilms.
31. Lawrence, J. R.,, G. M. Wolfaardt,, and D. R. Korber. 1994. Monitoring diffusion in biofilm matrices using scanning confocal laser microscopy. Appl. Environ. Microbiol. 60:11661173. The first application of 1P LSM to determine diffusion and penetration of size-fractionated probes in complex biofilms.
32. Lawrence, J. R.,, D. R. Korber,, G. M. Wolfaardt,, D. E. Caldwell, and T. R. Neu,. 2001. Analytical imaging and microscopy techniques, p. 3961. In C. J. Hurst,, R. L. Crawford,, G. R. Knudsen,, M. J. McInerney,, and L. D. Stetzenbach (ed.), Manual of Environmental Microbiology, 2nd ed. ASM Press, Washington, D.C. Comprehensive overview of digital microscopy as applied to a variety of environmental samples.
33. Lawrence, J. R.,, D. R. Korber,, B. D. Hoyle,, J. W. Costerton,, and D. E. Caldwell. 1991. Optical sectioning of microbial biofilms. J. Bacteriol. 173:65586567. The first comprehensive study of biofilm architecture applying 1P LSM and digital image analyses.
34. Lisle, J. T.,, P. S. Stewart,, and G. A. McFeters. 1999. Fluorescent probes applied to physiological characterization of bacterial biofilms. Methods Enzymol. 310:166178. A comprehensive overview with practical protocols for application of a number of fluorescent reporters to microorganisms.
35. Liss, S. N.,, I. G. Droppo,, D. T. Flannigan,, and G. G. Leppard. 1996. Floc architecture in wastewater and natural riverine systems. Environ. Sci. Technol. 30:680686. A useful example of the correlative microscopy approach.
36. Liu, J.,, F. B. Dazzo,, O. Glagoleva,, B. Yu,, and A. K. Jain. 2001. CMEIAS: a computer-aided system for the image analysis of bacterial morphotypes in microbial communities. Microb. Ecol. 41:173194. Excellent publication providing information on ImageTool and examples of the application of digital imaging and software routines in microbial ecology.
37. Manz, W.,, G. Arp,, G. Schumann-Kindel,, U. Szewzky,, and J. Reitner. 2000. Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue. J. Microbiol. Methods 40:125134. Excellent coverage of deconvolution approaches, with examples and color illustrations using microbial specimens.
38. Manz, W.,, K. Wendt-Potthoff,, T. R. Neu,, U. Szewzyk,, and J. R. Lawrence. 1999. Phylogenetic composition, spatial structure, and dynamics of lotic bacterial biofilms investigated by fluorescent in situ hybridization and confocal laser scanning microscopy. Microb. Ecol. 37:225237.
39. Massol-Deya, A.,, J. Whallon,, R. F. Hickey,, and J. Tiedje. 1995. Channel structures in aerobic biofilms of fixed film reactors treating contaminated groundwater. Appl. Environ. Microbiol. 61:767777.
40. Michael, T.,, and C. M. Smith. 1995. Lectins probe molecular films in biofouling: characterization of early films on non-living and living surfaces. Mar. Ecol. Progr. Ser. 119:229236. One of the first applications of lectins in complex habitats.
41. Møller, S.,, C. S. Kristensen,, L. K. Poulsen,, J. M. Carstensen,, and S. Molin. 1995. Bacterial growth on surfaces: automated image analysis for quantification of growth rate-related parameters. Appl. Environ. Microbiol. 61:741748.
42. Møller, S.,, D. R. Korber,, G. M. Wolfaardt,, S. Molin,, and D. E. Caldwell. 1997. The impact of nutrient composition on a degradative biofilm community. Appl. Environ. Microbiol. 63:24322438.
43. Neu, T. R. 2000. In situ cell and glycoconjugate distribution of river snow as studied by confocal laser scanning microscopy. Aquat. Microb. Ecol. 21:8595. Useful application publication illustrating information derived from 1P LSM based study.
44. Neu, T. R.,, G. D. W. Swerhone,, and J. R. Lawrence. 2001. Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147:299313. Excellent example of an assessment of fluors and probes for use with 1P LSM, with additional information on lectin applications.
45. Palmer, R., Jr.,, B. Applegate,, R. Burlage,, G. Sayle,r, and D. White,. 1998. Heterogeneity of gene expression and activity in bacterial biofilms, p. 609612. In A. Rhoda,, M. Pazzagli,, L. J. Kricka,, and P. E. Stanley (ed.), Bioluminescence and Chemical Luminescence: Perspectives for the 21st Century. Proceeding of the 10th International Symposium on Bioluminescence and Chemiluminescence. John Wiley and Sons, Chichester, United Kingdom. Useful example of correlative microscopy.
46. Pawley, J. B. (ed.). 2006. Handbook of Biological Confocal Microscopy. Plenum Press, New York, N.Y. An excellent technical summary of all aspects of confocal microscopy.
47. Perret, D.,, G. G. Leppard,, M. Muller,, N. Belzile,, R. DeVitre,, and R. Buffle. 1991. Electron microscopy of aquatic colloids: non-perturbing preparation of specimens in the field. Water Res. 25:13331343. Describes the use of Nanoplast embedding techniques for delicate microbial structures.
48. Podda, F.,, P. Zuddas,, A. Minacci,, M. Pepi,, and F. Baldi. 2000. Heavy metal coprecipitation with hydrozincite [Zn5(CO3)2(OH)6] from mine waters caused by photosynthetic microorganisms. Appl. Environ. Microbiol. 66:50925098.
49. Rocheleau, S.,, C. W. Greer,, J. R. Lawrence,, C. Cantin,, L. Laramee,, and S. Guiot. 1999. Differentiation of Methanosaeta concilii and Methanosarcina barkeri in anaerobic mesophilic granular sludge by fluorescent in situ hybridization and confocal scanning laser microscopy. Appl. Environ. Microbiol. 65:22222229. Excellent example of combined application of embedding, rRNA probes, 1P LSM, and digital image analyses.
50. Russ, J. C. (ed.). 2002. The Image Processing Handbook. CRC Press, Boca Raton, FL. Excellent comprehensive and understandable publication on all aspects of image processing.
51. Sanders, R.,, A. Draaijer,, H. C. Gerritsen,, P. M. Houpt,, and Y. K. Levine. 1995. Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. Anal. Biochem. 227:302308. Publication provides a basis for starting fluorescence lifetime imaging using 1P LSM systems.
52. Sytsma, J.,, J. M. Vroom,, C. J. de Grauw,, and H. C. Gerritsen. 1998. Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation. J. Microsc. 191:3951. Provides current application of 2P LSM in conjunction with fluorescence lifetime imaging.
53. Szmacinski, H.,, and J. R. Lakowicz. 1993. Optical measurements of pH using fluorescent lifetimes and phase-modulation fluorometry. Anal. Chem. 65:16681674. Excellent starting point for understanding application of fluorescence life-time imaging.
54. Ulrich, S.,, B. Karrasch,, H. G. Hoppe,, K. Jeskulke,, and M. Mehrens. 1996. Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl. Environ. Microbiol. 62:45874593. Good critical evaluation of the application of fluorescent probes to assess bacterial metabolism.
55. Valkenburg, J. A.,, C. L. Woldringh,, G. J. Brakenhoff,, H. T. van der Voort,, and N. Nanninga. 1985. Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images. J. Bacteriol. 161:478483. First application of confocal 1P LSM to study bacterial structure.
56. Van Ommen Kloeke, F.,, and G. G. Geesey. 1999. Localization and identification of populations of phosphatase- active bacterial cells associated with activated sludge flocs. Microb. Ecol. 38:201214. Example of the application of fluorescence imaging of alkaline phosphatase activity in flocs.
57. Vroom, J. M.,, K. J. de Grauw,, H. C. Gerritsen,, D. J. Bradshaw,, P. D. Marsh,, G. K. Watson,, J. J. Birmingham,, and C. Allison. 1999. Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy. Appl. Environ. Microbiol. 65:35023511. First substantial publication describing the use of 2P LSM to image microbial biofilms.
57.a. Wigglesworth-Cooksey, B.,, and K. E. Cooksey. 2005. Use of fluorophore-conjugated lectins to study cell-cell interactions in model marine biofilms. Appl. Environ. Microbiol. 71:428435. A good example of more extensive applications of lectins in marine habitats.
58. Wiggli, M.,, A. Smallcombe,, and R. Bachofen. 1999. Reflectance spectroscopy and laser confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine bog pond. J. Microbiol. Meth. 34:173182. Useful example of LSM imaging using reflectance, autofluorescence, and staining to examine environmental samples.
59. Wolfaardt, G. M.,, J. R. Lawrence,, R. D. Robarts,, and D. E. Caldwell. 1998. In situ characterization of biofilm exopolymers involved in the accumulation of chlorinated organics. Microb. Ecol. 35:213223. Application of fluor-conjugated lectins in complex biofilm system with quantification and analyses.
60. Wuertz, S.,, E. Muller,, R. Spaeth,, P. Pfleiderer,, and H.-C. Flemming. 2000. Detection of heavy metals in bacterial biofilms and microbial flocs with the fluorescent complexing agent Newport Green. J. Ind. Microbiol. Biotechnol. 24:116123. A good practical example of the application of LSM and calibration of fluorescence imaging.
61. Xiao, G. Q.,, and G. S. Kino. 1987. A real time confocal scanning optical microscope. Proc. Soc. Photo Opt. Instrum. Eng. 809:107113. Presents a useful, simple equation for estimation of optical section thickness.
62. Yu, F. P.,, G. M. Callis,, P. S. Stewart,, T. Griebe,, and G. A. McFeters. 1994. Cryosectioning of biofilms for microscopic examination. Biofouling 8:8591. An early example of this now-standard method.

Tables

Generic image for table
TABLE 1

Advantages and disadvantages of CLSM

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
Generic image for table
TABLE 2

Advantages and disadvantages of 2P LSM

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
Generic image for table
TABLE 3

Fluorescent compounds and their application in microbial studies

Numbers in parentheses are excitation maximums in nanometers.

FISH-MAR, FISH microautoradiography.

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3
Generic image for table
TABLE 4

Methods for the presentation of 3-D data sets

Citation: Lawrence J, Neu T. 2007. Laser Scanning Microscopy, p 34-53. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch3

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