3D imaging of biofilms on implants by detection of scattered light with a scanning laser optical tomograph

Marko Heidrich,Mark P Kühnel,Tineke Lange,Heiko Meyer,Meike Stiesch,Manuela Kellner,Raoul-Amadeus Lorbeer,Andreas Winkel,Alexander Heisterkamp

doi:10.1364/boe.2.002982

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.

Highlights

Bacteria and other microorganisms like fungi, algae and protozoa are able to form interfaceattached sessile communities of multispecies diversity embedded within a matrix of extracellular polymeric substances (EPS)
scanning laser optical tomography (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
The agarose appears two times brighter in the green channel than in the red channel and the signal generated by scattering at the metal surface itself is nearly independent of the used illumination laser, as expected

Summary

Introduction

Bacteria and other microorganisms like fungi, algae and protozoa are able to form interfaceattached sessile communities of multispecies diversity embedded within a matrix of extracellular polymeric substances (EPS) These so-called biofilms are ubiquitous and appear frequently resistant against host immune defense or medical treatments due to the combination of different bacterial characteristics and multiple functions of protective matrices [1]. For the investigation of this range, termed mesoscale, two techniques have been applied so far: Magnetic resonance microscopy (MRM), which is magnetic resonance imaging (MRI) at microscopic level, resolves biofilm structures with an isotropic resolution (< 100 μm, rarely less than 10 μm, [9]) and allows sample sizes of several millimeters in diameter [10, 11, 12, 13]. Detection and quantification of biofilm in the middle ear was demonstrated by the application of low coherence interferometry (LCI) in vivo [18]

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Discussion

Conclusion