Abstract

Biofilms – communities of microorganisms attached to surfaces – are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far.

Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.

© 2011 OSA

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2011

2010

R. Lorbeer, H. Meyer, M. Heidrich, H. Lubatschowski, and A. Heisterkamp, “Applying optical Fourier filtering to standard optical projection tomography,” Proc. SPIE7570, 75700 (2010).
[CrossRef]

U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, “Correction for specimen movement and rotation errors for in-vivo optical projection tomography,” Biomed. Opt. Express1, 87–96 (2010).
[CrossRef]

C. T. Nguyen, H. Tu, E. J. Chaney, C. N. Stewart, and S. A. Boppart, “Non-invasive optical interferometry for the assessment of biofilm growth in the middle ear,” Biomed. Opt. Express1, 1104–1116 (2010).
[CrossRef]

T. R. Neu, B. Manz, F. Volke, J. J. Dynes, A. P. Hitchcock, and J. R. Lawrence, “Advanced imaging techniques for assessment of structure, composition and function in biofilm systems,” FEMS Microbiol. Ecol.72, 1–21 (2010).
[CrossRef] [PubMed]

M. Wagner, D. Taherzadeh, C. Haisch, and H. Horn, “Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography,” Biotechnol. Bioeng.107, 844–853 (2010).
[CrossRef] [PubMed]

2009

E. Morgenroth and K. Milferstedt, “Biofilm engineering: linking biofilm development at different length and time scales,” Rev. Environ. Sci. Bio/Technology8, 203–208 (2009).
[CrossRef]

K. Subramani, R. E. Jung, A. Molenberg, and C. H. F. Hammerle, “Biofilm on dental implants: a review of the literature,” Int. J. Oral Maxillofacial Implants24, 616–626 (2009).

2008

H. Meyer, A. Darrell, A. Metaxakis, C. Savakis, and J. Ripoll, “Optical projection tomography for in-vivo imaging of drosophila melanogaster,” Microsc. Anal.22, 19–22 (2008).

N. U. Zitzmann and T. Berglundh, “Definition and prevalence of peri-implant diseases,” J. Clin. Periodontol.35, 286–291 (2008).
[CrossRef] [PubMed]

2007

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
[CrossRef]

J. S. McLean, O. N. Ona, and P. D. Majors, “Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy,” ISME J2, 121–131 (2007).
[CrossRef]

C. Haisch and R. Niessner, “Visualisation of transient processes in biofilms by optical coherence tomography,” Water Res.41, 2467–2472 (2007).
[CrossRef] [PubMed]

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Resolution improvement in emission optical projection tomography,” Phys. Med. Biology52, 2775–2790 (2007).
[CrossRef]

H. Meyer, A. Garofalakis, G. Zacharakis, S. Psycharakis, C. Mamalaki, D. Kioussis, E. N. Economou, V. Ntziachristos, and J. Ripoll, “Noncontact optical imaging in mice with full angular coverage and automatic surface extraction,” Appl. Opt.46, 3617–3627 (2007).
[CrossRef] [PubMed]

2006

S. Bolte and F. P. Cordelières, “A guided tour into subcellular colocalization analysis in light microscopy,” J. Microsc.224, 213–232 (2006).
[CrossRef]

H. Daims, S. Lücker, and M. Wagner, “daime, a novel image analysis program for microbial ecology and biofilm research,” Environ. Microbiol.8, 200–213 (2006).
[CrossRef] [PubMed]

M. Burmølle, J. S. Webb, D. Rao, L. H. Hansen, S. J. Sørensen, and S. Kjelleberg, “Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms,” Appl. Environ. Microbiol.72, 3916–3923 (2006).
[CrossRef] [PubMed]

C. Xi, D. Marks, S. Schlachter, W. Luo, and S. A. Boppart, “High-resolution three-dimensional imaging of biofilm development using optical coherence tomography,” J. Biomed. Opt.11, 034001 (2006).
[CrossRef]

2005

J. M. Tyszka, S. E. Fraser, and R. E. Jacobs, “Magnetic resonance microscopy: recent advances and applications,” Curr. Opinion Biotechnol.16, 93–99 (2005).
[CrossRef]

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Correction of artefacts in optical projection tomography,” Phys. Med. Biol.50, 4645–4665 (2005).
[CrossRef] [PubMed]

2004

H. Beyenal, C. Donovan, Z. Lewandowski, and G. Harkin, “Three-dimensional biofilm structure quantification,” J. Microbiol. Methods59, 395–413 (2004).
[CrossRef] [PubMed]

J. D. Seymour, S. L. Codd, E. L. Gjersing, and P. S. Stewart, “Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor,” J. Magn. Reson.167, 322–327 (2004).
[CrossRef] [PubMed]

2003

B. Manz, F. Volke, D. Goll, and H. Horn, “Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI),” Biotechnol. Bioeng.84, 424–432 (2003).
[CrossRef] [PubMed]

2002

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

J. Gabrielson, M. Hart, A. Jarelöv, I. Kühn, D. McKenzie, and R. Möllby, “Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates,” J. Microbiol. Methods50, 63–73 (2002).
[CrossRef] [PubMed]

2000

T. Beikler and T. F. Flemmig, “Oral biofilm-associated diseases: trends and implications for quality of life, systemic health and expenditures,” Periodontology200055, 87–103 (2011).
[CrossRef]

A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
[PubMed]

1998

M. Kuehn, M. Hausner, H. Bungartz, M. Wagner, P. A. Wilderer, and S. Wuertz, “Automated confocal laser scanning microscopy and semiautomated image processing for analysis of biofilms,” Appl. Environ. Microbiol.64, 4115–4127 (1998).
[PubMed]

1993

Z. Lewandowski, S. A. Altobelli, and E. Fukushima, “NMR and microelectrode studies of hydrodynamics and kinetics in biofilms,” Biotechnol. Prog.9, 40–45 (1993).
[CrossRef]

1991

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
[CrossRef] [PubMed]

J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
[PubMed]

1976

1967

R. P. Tengerdy, J. G. Nagy, and B. Martin, “Quantitative measurement of bacterial growth by the reduction of tetrazolium salts,” Appl. Microbiol.15, 954–955 (1967).
[PubMed]

P. L. Steponkus and F. O. Lanphear, “Refinement of the triphenyl tetrazolium chloride method of determining cold injury,” Plant Physiol.42, 1423 –1426 (1967).
[CrossRef] [PubMed]

1949

E. Kun and L. G. Abood, “Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride,” Science109, 144 –146 (1949).
[CrossRef] [PubMed]

1941

R. Kuhn and D. Jerchel, “Über Invertseifen, VIII. mitteil.: Reduktion von Tetrazoliumsalzen durch Bakterien, gärende Hefe und keimende Samen,” Ber. Dtsch. Chem. Ges. A B74, 949–952 (1941).
[CrossRef]

Abood, L. G.

E. Kun and L. G. Abood, “Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride,” Science109, 144 –146 (1949).
[CrossRef] [PubMed]

Ahlgren, U.

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

Altobelli, S. A.

Z. Lewandowski, S. A. Altobelli, and E. Fukushima, “NMR and microelectrode studies of hydrodynamics and kinetics in biofilms,” Biotechnol. Prog.9, 40–45 (1993).
[CrossRef]

Anderson, G. G.

G. G. Anderson and G. A. O’Toole, “Innate and induced resistance mechanisms of bacterial biofilms,” Curr. Top. Microbiol. Immunol.322, 85–105 (2008).
[CrossRef] [PubMed]

Baldock, R.

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

Battin, T. J.

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
[CrossRef]

Beikler, T.

T. Beikler and T. F. Flemmig, “Oral biofilm-associated diseases: trends and implications for quality of life, systemic health and expenditures,” Periodontology200055, 87–103 (2011).
[CrossRef]

Berglundh, T.

N. U. Zitzmann and T. Berglundh, “Definition and prevalence of peri-implant diseases,” J. Clin. Periodontol.35, 286–291 (2008).
[CrossRef] [PubMed]

Beyenal, H.

H. Beyenal, C. Donovan, Z. Lewandowski, and G. Harkin, “Three-dimensional biofilm structure quantification,” J. Microbiol. Methods59, 395–413 (2004).
[CrossRef] [PubMed]

Bicker, G.

Birk, U. J.

Bolte, S.

S. Bolte and F. P. Cordelières, “A guided tour into subcellular colocalization analysis in light microscopy,” J. Microsc.224, 213–232 (2006).
[CrossRef]

Boppart, S. A.

C. T. Nguyen, H. Tu, E. J. Chaney, C. N. Stewart, and S. A. Boppart, “Non-invasive optical interferometry for the assessment of biofilm growth in the middle ear,” Biomed. Opt. Express1, 1104–1116 (2010).
[CrossRef]

C. Xi, D. Marks, S. Schlachter, W. Luo, and S. A. Boppart, “High-resolution three-dimensional imaging of biofilm development using optical coherence tomography,” J. Biomed. Opt.11, 034001 (2006).
[CrossRef]

Bugeon, L.

Bungartz, H.

M. Kuehn, M. Hausner, H. Bungartz, M. Wagner, P. A. Wilderer, and S. Wuertz, “Automated confocal laser scanning microscopy and semiautomated image processing for analysis of biofilms,” Appl. Environ. Microbiol.64, 4115–4127 (1998).
[PubMed]

Burmølle, M.

M. Burmølle, J. S. Webb, D. Rao, L. H. Hansen, S. J. Sørensen, and S. Kjelleberg, “Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms,” Appl. Environ. Microbiol.72, 3916–3923 (2006).
[CrossRef] [PubMed]

Caldwell, D. E.

J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
[PubMed]

Chaney, E. J.

Chang, W.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
[CrossRef] [PubMed]

Chen, L.

Codd, S. L.

J. D. Seymour, S. L. Codd, E. L. Gjersing, and P. S. Stewart, “Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor,” J. Magn. Reson.167, 322–327 (2004).
[CrossRef] [PubMed]

Cordelières, F. P.

S. Bolte and F. P. Cordelières, “A guided tour into subcellular colocalization analysis in light microscopy,” J. Microsc.224, 213–232 (2006).
[CrossRef]

Costerton, J. W.

J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
[PubMed]

Curtis, T. P.

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
[CrossRef]

Daims, H.

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
[CrossRef]

H. Daims, S. Lücker, and M. Wagner, “daime, a novel image analysis program for microbial ecology and biofilm research,” Environ. Microbiol.8, 200–213 (2006).
[CrossRef] [PubMed]

Dallman, M. J.

Darrell, A.

U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, “Correction for specimen movement and rotation errors for in-vivo optical projection tomography,” Biomed. Opt. Express1, 87–96 (2010).
[CrossRef]

H. Meyer, A. Darrell, A. Metaxakis, C. Savakis, and J. Ripoll, “Optical projection tomography for in-vivo imaging of drosophila melanogaster,” Microsc. Anal.22, 19–22 (2008).

Davidson, D.

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

Donovan, C.

H. Beyenal, C. Donovan, Z. Lewandowski, and G. Harkin, “Three-dimensional biofilm structure quantification,” J. Microbiol. Methods59, 395–413 (2004).
[CrossRef] [PubMed]

Drexler, W.

W. Drexler and J. G. Fujimoto, Optical Coherence Tomography: Technology and Applications (Springer, New York, 2008).
[CrossRef]

Dynes, J. J.

T. R. Neu, B. Manz, F. Volke, J. J. Dynes, A. P. Hitchcock, and J. R. Lawrence, “Advanced imaging techniques for assessment of structure, composition and function in biofilm systems,” FEMS Microbiol. Ecol.72, 1–21 (2010).
[CrossRef] [PubMed]

Eberl, L.

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
[CrossRef]

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D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
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Givskov, M.

A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
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J. D. Seymour, S. L. Codd, E. L. Gjersing, and P. S. Stewart, “Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor,” J. Magn. Reson.167, 322–327 (2004).
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B. Manz, F. Volke, D. Goll, and H. Horn, “Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI),” Biotechnol. Bioeng.84, 424–432 (2003).
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Gregory, K.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
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M. Wagner, D. Taherzadeh, C. Haisch, and H. Horn, “Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography,” Biotechnol. Bioeng.107, 844–853 (2010).
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K. Subramani, R. E. Jung, A. Molenberg, and C. H. F. Hammerle, “Biofilm on dental implants: a review of the literature,” Int. J. Oral Maxillofacial Implants24, 616–626 (2009).

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M. Burmølle, J. S. Webb, D. Rao, L. H. Hansen, S. J. Sørensen, and S. Kjelleberg, “Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms,” Appl. Environ. Microbiol.72, 3916–3923 (2006).
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T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
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J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
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D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
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R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. Ramírez Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, “Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph,” Opt. Express19, 5419–5430 (2011).
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R. Lorbeer, H. Meyer, M. Heidrich, H. Lubatschowski, and A. Heisterkamp, “Applying optical Fourier filtering to standard optical projection tomography,” Proc. SPIE7570, 75700 (2010).
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J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Resolution improvement in emission optical projection tomography,” Phys. Med. Biology52, 2775–2790 (2007).
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J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Correction of artefacts in optical projection tomography,” Phys. Med. Biol.50, 4645–4665 (2005).
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A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
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A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
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J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
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M. Wagner, D. Taherzadeh, C. Haisch, and H. Horn, “Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography,” Biotechnol. Bioeng.107, 844–853 (2010).
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B. Manz, F. Volke, D. Goll, and H. Horn, “Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI),” Biotechnol. Bioeng.84, 424–432 (2003).
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J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
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J. M. Tyszka, S. E. Fraser, and R. E. Jacobs, “Magnetic resonance microscopy: recent advances and applications,” Curr. Opinion Biotechnol.16, 93–99 (2005).
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J. Gabrielson, M. Hart, A. Jarelöv, I. Kühn, D. McKenzie, and R. Möllby, “Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates,” J. Microbiol. Methods50, 63–73 (2002).
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K. Subramani, R. E. Jung, A. Molenberg, and C. H. F. Hammerle, “Biofilm on dental implants: a review of the literature,” Int. J. Oral Maxillofacial Implants24, 616–626 (2009).

Kioussis, D.

Kjelleberg, S.

T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
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Korber, D. R.

J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
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M. Kuehn, M. Hausner, H. Bungartz, M. Wagner, P. A. Wilderer, and S. Wuertz, “Automated confocal laser scanning microscopy and semiautomated image processing for analysis of biofilms,” Appl. Environ. Microbiol.64, 4115–4127 (1998).
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R. Kuhn and D. Jerchel, “Über Invertseifen, VIII. mitteil.: Reduktion von Tetrazoliumsalzen durch Bakterien, gärende Hefe und keimende Samen,” Ber. Dtsch. Chem. Ges. A B74, 949–952 (1941).
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J. Gabrielson, M. Hart, A. Jarelöv, I. Kühn, D. McKenzie, and R. Möllby, “Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates,” J. Microbiol. Methods50, 63–73 (2002).
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J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton, and D. E. Caldwell, “Optical sectioning of microbial biofilms.” J. Bacteriol.173, 6558–6567 (1991).
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H. Beyenal, C. Donovan, Z. Lewandowski, and G. Harkin, “Three-dimensional biofilm structure quantification,” J. Microbiol. Methods59, 395–413 (2004).
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Z. Lewandowski, S. A. Altobelli, and E. Fukushima, “NMR and microelectrode studies of hydrodynamics and kinetics in biofilms,” Biotechnol. Prog.9, 40–45 (1993).
[CrossRef]

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D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
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Lorbeer, R.

R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. Ramírez Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, “Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph,” Opt. Express19, 5419–5430 (2011).
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R. Lorbeer, H. Meyer, M. Heidrich, H. Lubatschowski, and A. Heisterkamp, “Applying optical Fourier filtering to standard optical projection tomography,” Proc. SPIE7570, 75700 (2010).
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C. Xi, D. Marks, S. Schlachter, W. Luo, and S. A. Boppart, “High-resolution three-dimensional imaging of biofilm development using optical coherence tomography,” J. Biomed. Opt.11, 034001 (2006).
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J. S. McLean, O. N. Ona, and P. D. Majors, “Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy,” ISME J2, 121–131 (2007).
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Manz, B.

T. R. Neu, B. Manz, F. Volke, J. J. Dynes, A. P. Hitchcock, and J. R. Lawrence, “Advanced imaging techniques for assessment of structure, composition and function in biofilm systems,” FEMS Microbiol. Ecol.72, 1–21 (2010).
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B. Manz, F. Volke, D. Goll, and H. Horn, “Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI),” Biotechnol. Bioeng.84, 424–432 (2003).
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C. Xi, D. Marks, S. Schlachter, W. Luo, and S. A. Boppart, “High-resolution three-dimensional imaging of biofilm development using optical coherence tomography,” J. Biomed. Opt.11, 034001 (2006).
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McKenzie, D.

J. Gabrielson, M. Hart, A. Jarelöv, I. Kühn, D. McKenzie, and R. Möllby, “Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates,” J. Microbiol. Methods50, 63–73 (2002).
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J. S. McLean, O. N. Ona, and P. D. Majors, “Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy,” ISME J2, 121–131 (2007).
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H. Meyer, A. Darrell, A. Metaxakis, C. Savakis, and J. Ripoll, “Optical projection tomography for in-vivo imaging of drosophila melanogaster,” Microsc. Anal.22, 19–22 (2008).

Meyer, H.

R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. Ramírez Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, “Highly efficient 3D fluorescence microscopy with a scanning laser optical tomograph,” Opt. Express19, 5419–5430 (2011).
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R. Lorbeer, H. Meyer, M. Heidrich, H. Lubatschowski, and A. Heisterkamp, “Applying optical Fourier filtering to standard optical projection tomography,” Proc. SPIE7570, 75700 (2010).
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U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, “Correction for specimen movement and rotation errors for in-vivo optical projection tomography,” Biomed. Opt. Express1, 87–96 (2010).
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H. Meyer, A. Darrell, A. Metaxakis, C. Savakis, and J. Ripoll, “Optical projection tomography for in-vivo imaging of drosophila melanogaster,” Microsc. Anal.22, 19–22 (2008).

H. Meyer, A. Garofalakis, G. Zacharakis, S. Psycharakis, C. Mamalaki, D. Kioussis, E. N. Economou, V. Ntziachristos, and J. Ripoll, “Noncontact optical imaging in mice with full angular coverage and automatic surface extraction,” Appl. Opt.46, 3617–3627 (2007).
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K. Subramani, R. E. Jung, A. Molenberg, and C. H. F. Hammerle, “Biofilm on dental implants: a review of the literature,” Int. J. Oral Maxillofacial Implants24, 616–626 (2009).

Molin, S.

A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
[PubMed]

Möllby, R.

J. Gabrielson, M. Hart, A. Jarelöv, I. Kühn, D. McKenzie, and R. Möllby, “Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates,” J. Microbiol. Methods50, 63–73 (2002).
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E. Morgenroth and K. Milferstedt, “Biofilm engineering: linking biofilm development at different length and time scales,” Rev. Environ. Sci. Bio/Technology8, 203–208 (2009).
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R. P. Tengerdy, J. G. Nagy, and B. Martin, “Quantitative measurement of bacterial growth by the reduction of tetrazolium salts,” Appl. Microbiol.15, 954–955 (1967).
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T. R. Neu, B. Manz, F. Volke, J. J. Dynes, A. P. Hitchcock, and J. R. Lawrence, “Advanced imaging techniques for assessment of structure, composition and function in biofilm systems,” FEMS Microbiol. Ecol.72, 1–21 (2010).
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Nielsen, A. T.

A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, “Quantification of biofilm structures by the novel computer program COMSTAT,” Microbiology146, 2395 –2407 (2000).
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C. Haisch and R. Niessner, “Visualisation of transient processes in biofilms by optical coherence tomography,” Water Res.41, 2467–2472 (2007).
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O’Toole, G. A.

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J. S. McLean, O. N. Ona, and P. D. Majors, “Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy,” ISME J2, 121–131 (2007).
[CrossRef]

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J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
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Puliafito, C. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
[CrossRef] [PubMed]

Ramírez Ojeda, D. F.

Rao, D.

M. Burmølle, J. S. Webb, D. Rao, L. H. Hansen, S. J. Sørensen, and S. Kjelleberg, “Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms,” Appl. Environ. Microbiol.72, 3916–3923 (2006).
[CrossRef] [PubMed]

Rieckher, M.

Ripoll, J.

Ross, A.

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

Sarasa-Renedo, A.

Savakis, C.

H. Meyer, A. Darrell, A. Metaxakis, C. Savakis, and J. Ripoll, “Optical projection tomography for in-vivo imaging of drosophila melanogaster,” Microsc. Anal.22, 19–22 (2008).

Schlachter, S.

C. Xi, D. Marks, S. Schlachter, W. Luo, and S. A. Boppart, “High-resolution three-dimensional imaging of biofilm development using optical coherence tomography,” J. Biomed. Opt.11, 034001 (2006).
[CrossRef]

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D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, “Optical coherence tomography,” Science254, 1178 –1181 (1991).
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J. D. Seymour, S. L. Codd, E. L. Gjersing, and P. S. Stewart, “Magnetic resonance microscopy of biofilm structure and impact on transport in a capillary bioreactor,” J. Magn. Reson.167, 322–327 (2004).
[CrossRef] [PubMed]

Sharpe, J.

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Resolution improvement in emission optical projection tomography,” Phys. Med. Biology52, 2775–2790 (2007).
[CrossRef]

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Correction of artefacts in optical projection tomography,” Phys. Med. Biol.50, 4645–4665 (2005).
[CrossRef] [PubMed]

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science296, 541–545 (2002).
[CrossRef] [PubMed]

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J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Resolution improvement in emission optical projection tomography,” Phys. Med. Biology52, 2775–2790 (2007).
[CrossRef]

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, “Correction of artefacts in optical projection tomography,” Phys. Med. Biol.50, 4645–4665 (2005).
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T. J. Battin, W. T. Sloan, S. Kjelleberg, H. Daims, I. M. Head, T. P. Curtis, and L. Eberl, “Microbial landscapes: new paths to biofilm research,” Nat. Rev. Microbiol.5, 76–81 (2007).
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Supplementary Material (2)

» Media 1: MOV (2791 KB)     
» Media 2: MOV (3977 KB)     

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Figures (7)

Figure 1
Figure 1

SLOT setup. A removable mirror (rm) allows one to choose between two laser sources, a helium-neon laser and a green laser module, respectively. The spatially cleaned and aperture-adjusted laser beam is scanned through the sample which is mounted rotatably in a glass test tube positioned in a cuvette filled with water. Scattered laser light that is emitted to the bottom of the cuvette and reflected from its walls is focused onto a photomultiplier tube (PMT). Transmitted light is captured by a photodiode (PD). ph: pinhole; d: diaphragm; gm: x–y galvo mirror.

Figure 2
Figure 2

Prepared sample: A metal cylinder or dental implant is mounted within an agarose gel containing the redox indicator triphenyltetrazolium chloride (TTC) and a nutrient concentration gradient. A plastic tube centrically aligns the implant within the surrounding glass tube and contains the bacteria which are re-suspended in PBS. The setup allows the bacteria to migrate along the implant surface starting at the application side while having access to nutrients and TTC in the surrounding agarose gel. An adapter fixates the glass test tube with the rotation stage of the SLOT setup (Fig. 1).

Figure 3
Figure 3

Projection images of two metal cylinder samples covered with biofilm consisting of Bacillus cereus (a–d) and Pseudomonas stutzeri (e–h). The samples were illuminated with both laser sources (helium-neon laser: (a,e); green laser module: (b,f)). Each image was acquired by the detection of scattered laser light at the same viewing angle. Composite images (c,g) were generated by merging red and green illuminated parts. The calculation of the difference signal provides projection images showing the biofilm only (d,h). Scale bars represent 500 μm.

Figure 4
Figure 4

Projection images of a dental implant covered with biofilm consisting of Bacillus cereus. The samples were illuminated with both laser sources (helium-neon laser: (a); green laser module: (b)). Each image was acquired by the detection of scattered laser light at the same viewing angle. (c) is the composite image ( Media 1) generated by merging (a) and (b) while the difference signal image is shown in (d). Scale bars represent 500 μm.

Figure 5
Figure 5

Reconstructed and processed volumetric stacks of the biofilm raw data shown in Fig. 3h: (a) Rendered image of the biofilm (represented in red) and the surface of the metal cylinder (gray). Difference signal data set containing the biofilm distribution and transmission data set were used for reconstruction. (b) Slice through the volumetric data stack of the biofilm along the curved cylindrical metal surface. This corresponds to a transformation of the curved cylindrical coordinates into a flat Euclidean plane. Scale bars represent 500 μm.

Figure 6
Figure 6

Reconstructed and processed volumetric stacks of the biofilm raw data shown in Fig. 4d. The difference signal data set containing the biofilm distribution was used for reconstruction. (a) Rendered image of the biofilm (represented in red) ( Media 2). (b) Radial maximum intensity projection (rMIP) of the volumetric data stack of the biofilm. Scale bars represent 500 μm.

Figure 7
Figure 7

Analysis of biofilm clusters on a titanium cylinder at two points in time: Maximum intensity projection (left) and cluster volume as a function of height position on the implant surface (right) of reconstructed biofilm two days after preparation (a,b) and 24 h later (c,d). In accordance with the nutrient concentration gradient in the agarose gel, the cluster volume in the upper region is larger than in the lower region of the sample. The cluster number increases from 246 to 467 within 24 h. Biofilm: Extraction of patient’s dental plaque. Scale bars represent 500 μm.

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