Langmuir 2001, 17, 2235-2242
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Nanometer Distances between Swimming Bacteria and Surfaces Measured by Total Internal Reflection Aqueous Fluorescence Microscopy Margot A.-S. Vigeant,*,† Michael Wagner,‡ Lukas K. Tamm,‡ and Roseanne M. Ford† Department of Chemical Engineering, School of Engineering and Applied Science, and Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, Virginia 22904-4741 Received August 31, 2000. In Final Form: December 21, 2000 Bacterial adhesion to surfaces can lead to the formation of biofilms and the development of infection. Bacteria which are motile reach surfaces faster than nonmotile bacteria and may adhere more rapidly than nonmotile bacteria. The motility of a species has also been implicated as a factor in virulence. Understanding the role that motility plays in bringing a bacterium in contact with a surface and its subsequent adherence can aid in designing strategies to prevent adhesion. In this paper, we describe the development of a total internal reflection aqueous fluorescence (TIRAF) microscope to measure the distance between an E. coli bacterium and a clean quartz surface as the bacterium was swimming laterally along the surface. This technique is distinct from other related approaches such as atomic force microscopy and total internal reflection fluorescence microscopy because it does not require the immobilization of cells on a surface for the measurement. The TIRAF microscope was capable of capturing images of a field of bacteria two times per second for over 1 min. The analysis technique developed to translate the images into quantitative distance measurements, using the equations of Gingell, is also described. Both motile and nonmotile bacteria were observed within 100 nm of a clean quartz surface. TIRAF provided a quantitative measure of the distance between bacteria and a surface at nanometer scale resolution.
Introduction Bacterial interactions with surfaces affect our lives in many different ways. In some cases the attachment to a surface is desirable (as with biofilters which are used to remove contaminants in groundwater1) while in other cases it may prove to be detrimental (as in the case of dental caries). To control the formation of biofilms in preventing disease or enhancing the remediation of polluted groundwater, it is important to understand the events which immediately precede the permanent attachment of these microorganisms to surfaces.2,3 Motile bacteria such as E. coli move in a manner resembling a random walk when in bulk fluid.4 However, when these cells come close to a surface, the cells will often turn and swim parallel to that surface for some time.5-9 Cells which swim along the surface may then * Corresponding author: Dr. Margot A.-S. Vigeant, Department of Chemical Engineering, Bucknell University, Lewisburg, PA 17837. Phone: (570) 577-1646. Fax: (570) 577-1141. E-mail:
[email protected]. † Department of Chemical Engineering, School of Engineering and Applied Science. ‡ Department of Molecular Physiology and Biological Physics, School of Medicine. (1) Shareefdeen, Z.; Baltzis, B. C.; Oh, Y.-S.; Bartha, R. Biotechnol. Bioeng. 1993, 41, 512-524. (2) Fletcher, M., Savage, D. C., Eds. Bacterial Adhesion: Mechanisms and Physiological Significance; Plenum Press: New York, 1985. (3) Fletcher, M., Ed. Bacterial Adhesion: Molecular and Ecological Diversity; Weily-Liss: New York, 1996. (4) Berg, H. C. Random Walks in Biology; Princeton University Press: Princeton, NJ, 1993. (5) Berg, H. C.; Turner, L. Biophys. J. 1990, 58, 919-930. (6) Frymier, P. D.; Ford, R. M.; Cummings, P. T. Chem. Eng. Sci. 1993, 48, 687-699. (7) Lawrence, J. R.; Delaquis, P. J.; Korber, D. R.; Caldwell, D. E. Microb. Ecol. 1987, 14, 1-14. (8) Maeda, K.; Imae, Y.; Shioi, J.-I.; Oosawa, F. J. Bacteriol. 1976, 127, 1039-1046.
become loosely attached to the surface.7 This is the first step in the process of biofilm formation, as described by Marshall.10 In this loose attachment, cells will continue to exhibit Brownian motion. Bacteria may then proceed to a more firm, irreversible attachment. To complete the biofilm formation process, adherent cells will divide, producing more cells, and may also recruit other bacteria from the bulk fluid to adhere to the surface. Motile bacteria progress through these initial steps more quickly than nonmotile bacteria, especially under conditions where fluid flow is significant.11 We were interested in determining the distance from the surface at which the bacteria swim along and become loosely attached to the surface, so that we may determine the forces responsible for the initial steps of adhesion. To determine this distance, we have developed a total internal reflection aqueous fluorescence (TIRAF) microscopy system to quantitatively assess the distance between a bacterium and a surface. There are a number of techniques currently in use for the measurement of forces or distances between bacteria (or model cells) and surfaces. These are atomic force microscopy (AFM),12-14 the surface forces apparatus (SFA),15-19 and total internal reflection microscopy (TIRM).20-23 The main reason that each of these methods (9) Vigeant, M. A.-S.; Ford, R. M. Appl. Environ. Microbiol. 1997, 63, 3474-3479. (10) Marshall, K. C.; Stout, R.; Mitchell, R. J. Gen. Microbiol. 1971, 68, 337-348. (11) Korber, D. R.; Lawrence, J. R.; Sutton, B.; Caldwell, D. E. Microb. Ecol. 1989, 18, 1-19. (12) Camesano, T. A.; Logan, B. E. Environ. Sci. Technol., in press. (13) Razatos, A.; Ong, Y. L.; Sharma, M.; Georgiou, G. J. Biomater. Sci., Polym. Ed. 1998, 9, 1362-1373. (14) Razatos, A.; Ong, Y.-L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11059-11064. (15) Kuhl, T.; Guo, Y.; Alderfer, J.; Berman, A.; Leckband, D.; Israelachvili, J.; Hui, S. Langmuir 1996, 12, 3003-3014.
10.1021/la0012539 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/08/2001
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is not suitable for observing cells while they move laterally along a surface is that all of these methods, in one way or another, constrain the motion of swimming of cells. With TIRAF microscopy, quantitative surface separation distances can be measured for motile cells, as long as the refractive indices of the cells and the medium are known. Both TIRAF and the more standard TIRF (total internal reflection fluorescence) microscopy utilize the same basic apparatus and illumination through total internal reflection. However, TIRF requires that cells be labeled fluorescently or be intrinsically fluorescent,24 while in TIRAF the fluorescent label is in the aqueous phase.25 One advantage of TIRAF over TIRF is that by not labeling the cell itself, the concern of whether or not attaching a fluorophore changes the surface properties of the cell is eliminated. In TIRF all cells would have to be of known size and be labeled with identical (known) amounts and distributions of fluorophore in order to make quantitative measurements of the distance. TIRAF avoids this problem because the aqueous concentration of fluorophore is much easier to control precisely. In this work we describe the application of TIRAF microscopy for measuring the distance between a bacterium and a clean quartz surface as the bacterium adheres to or swims laterally along the surface. Images of bacteria near the surface and details of the novel analysis used to obtain quantitative measures of the distance are included. As an example of measurements that were obtained with TIRAF, the distribution of distances is compared for a nonmotile strain of bacteria and a smooth-swimming strain. Finally, we discuss several issues related to the accuracy of this technique. Background TIRAF microscopy was first described by Gingell,25-29 and has also been recently applied by Geggier and Fuhr to study L929 fibroblast cells.30 TIRAF belongs to a family of microscopy techniques including TIRF and TIRM which derive their illumination from the evanescent wave created when light is totally reflected from an interface. The generation of such a wave is illustrated in Figure 1. When a beam of light is incident from a medium of high refractive index to one of lower refractive index at an angle above the critical angle θc, total internal reflection occurs. Although all of the visible light is reflected from the (16) Leckband, D.; Mueller, W.; Schmitt, F. J.; Ringsdorf, H. Biophys. J. 1995, 69, 1162-1169. (17) Sheth, S.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8399-8404. (18) Yu, Z.; Calvert, T.; Leckband, D. Biochemistry 1998, 37, 15401550. (19) Wong, J.; Kuhl, T.; Israelachvili, J.; Mullah, N.; Zalipsky, S. Science 1997, 275, 820-822. (20) Robertson, S. K.; Uhrick, A. F.; Bike, S. G. J. Colloid Interface Sci. 1998, 202, 208-211. (21) Robertson, S. K.; Bike, S. G. Langmuir 1998, 14, 928-934. (22) Clapp, A. R.; Ruta, A. G.; Dickinson, R. B. Rev. Sci. Instrum. 1999, 70, 2627-2636. (23) Truesdail, S.; Clapp, A.; Shah, D.; Dickinson, R. Fundamental and Applied Aspects of Chemically Modified Surfaces. In Symposium on Chemically Modified Surfaces; Royal Society of Chemistry: Cambridge, 1999; Vol. 235, pp 369-378. (24) Burmeister, J. S.; Olivier, L. A.; Reichert, W. M.; Truskey, G. A. Biomaterials 1998, 19, 307-325. (25) Gingell, D.; Todd, I.; Bailey, J. J. Cell Biol. 1985, 100, 13341338. (26) Gingell, D.; Parsegian, V. A. J. Theor. Biol. 1972, 36, 41-52. (27) Gingell, D.; Heavens, O. J. Microsc. 1996, 182, 141-148. (28) Gingell, D.; Heavens, O. S.; Mellor, J. S. J. Cell Sci. 1987, 87, 677-693. (29) Mellor, J. S.; Gingell, D.; Heavens, O. S. J. Mod. Opt. 1988, 35, 623-628. (30) Geggier, P.; Fuhr, G. Appl. Phys. A 1999, 68, 505-513.
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Figure 1. Illustration of the creation of an evanescent wave. A laser beam (above, left) strikes the quartz/water interface at an angle φ, greater than the critical angle. The beam is totally reflected (above, right), but creates an evanescent wave in the liquid medium. This wave, which decreases exponentially with distance from the surface, can excite fluorescence in fluoresceindextran molecules in the aqueous solution. Adapted from ref 37.
Figure 2. Evanescent wave field strength (a) in the absence of and (b) in the presence of a bacterium. The intensity of light seen by an observer is proportional to the shaded area under the curve. Reprinted with permission from ref 27. Copyright 1996 Blackwell.
interface, electromagnetic energy is carried across the interface in the form of an evanescent wave, which follows an exponential decay with depth from the interface, falling to undetectable levels within less than one wavelength. Because the evanescent wave changes in a predictable way with distance from the interface, it is especially useful for measuring small (
