Quantitative 3D comparison of biofilm imaged by X‐ray micro‐tomography and two‐photon laser scanning microscopy

Summary Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X‐ray micro‐tomography (X‐ray CMT) might be an alternative but it finds limitations in similarity of X‐ray absorption coefficien...

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Bibliographic Details
Published in:Journal of microscopy (Oxford) 2018-09, Vol.271 (3), p.302-314
Main Authors: LARUE, A.E., SWIDER, P., DURU, P., DAVIAUD, D., QUINTARD, M., DAVIT, Y.
Format: Article
Language:English
Subjects:
3D printing
Absorptivity
Barite
Barium
Barium sulfate
Beads
Bias
Biofilms
Biomechanics
Capillaries
Carbon dioxide
Carbon sequestration
Confocal microscopy
contrast agent
Contrast agents
Dispersion
Engineering Sciences
Enhanced oil recovery
Fluid mechanics
Fluorescence
Gelation
Hysteresis
Image processing
Image segmentation
Imaging techniques
Lasers
Mechanics
Media
Medical imaging
Microorganisms
Microscopy
Microtomography
Physics
Porous materials
Porous media
Pseudomonas aeruginosa
Quantitative analysis
Sedimentation
Sedimentation & deposition
Soil remediation
Sulfates
Surface area
Three dimensional imaging
Tomography
two‐photon microscopy
X‐ray micro‐tomography
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Summary:Summary Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X‐ray micro‐tomography (X‐ray CMT) might be an alternative but it finds limitations in similarity of X‐ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high‐resolution 3D imaging of biofilm via X‐ray CMT. However, this approach lacks comparison with well‐established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two‐photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X‐ray CMT using an improved protocol where barium sulphate is combined with low‐gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono‐dispersed fluorescent and X‐ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X‐ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM. Lay description Bacteria often develop in sessile colonies, termed biofilms, where the micro‐organisms are embedded in a complex self‐secreted polymer matrix. Biofilms developing in porous media are a key process to many engineering applications, for example bio‐filters, soil bio‐remediation, CO2 storage and microbial enhanced oil recovery. When investigating biofilm development in porous media, 3D observation of the biofilm distribution is restricted due to the limits of currently used imaging methods. Confocal laser scanning and multi‐photon microscopy, which are widely used techniques for biofilms, are not applicable to a porous and generally opaque structure. Approaches based on X‐ray micro‐tomography (X‐ray CMT) are being explored as this technique theoretically enables the imaging of large volumes of porous media with submicron resolution. However, X‐ray absorption coefficients for the biofilm and the surrounding aqueous phase are ve
ISSN:0022-2720
1365-2818
DOI:10.1111/jmi.12718