Nonlinear laser scanning microscopy of oral multispecies-biofilms: fixative induced fluorescence as a fast and economical in vitro screening method

Andric, Nadine; Ehmke, Tobias; Stumpp, Nico; Ripken, Tammo; Heisterkamp, Alexander; Stiesch, Meike

doi:10.1515/bnm-2015-0028

Cited by 3 publications

(2 citation statements)

References 14 publications

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“…The urea-NaCl-FISH assay was first described for Staphylococcus aureus [ 37 ]. We have already used this method in a study on the simultaneous staining of three species [ 42 ]. Our results show that urea-NaCl-FISH assay is also suitable for simultaneous FISH analysis of four different gram-positive and gram-negative bacterial species embedded in a biofilm.…”

Section: Discussionmentioning

confidence: 99%

An oral multispecies biofilm model for high content screening applications

et al. 2017

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Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.

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Section: Discussionmentioning

confidence: 99%

An oral multispecies biofilm model for high content screening applications

et al. 2017

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“…5,6 Andric et al have shown TPEF measurements with fixated bacterial biofilms can distinguish species by their respective fluorescence intensity. 7 In animal, and especially immune cells, TPEF and FLIM are being used as a non-invasive way to distinguish cells. [8][9][10][11] FLIM extends the possibilities of TPEF by measuring the temporal component of the incoming signal and can be used to distinguish between the free and bound state of the coenzymes.…”

Section: Introductionmentioning

confidence: 99%

Multimodal imaging of bacterial species for label-free classification

Dyrøy,

Heitz,

Studier

et al. 2024

Label-Free Biomedical Imaging and Sensing (LBIS) 2024

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Bacterial infections often pose a time-sensitive concern; however, the current diagnostic methods are comprised of lengthy processes. This study explores label-free imaging techniques based on two-photon excitation of intrinsic molecules found in various bacterial species. Specifically, two-photon excitation microscopy (TPEF) and fluorescence lifetime imaging microscopy (FLIM) are employed. These methods have been extensively utilized in the area of animal cells, yielding favorable outcomes, yet their application to bacteria remains largely unexplored. Analogous to the work on animal cells, initial attention is directed towards the metabolic coenzymes, namely nicotinamide adenine dinucleotide (phosphate) (NADPH) and flavin adenine dinucleotide (FAD). The extra time component of the FLIM setup was used to further investigate differences between the decay curves of the emitted autofluorescence in their different growth phases, corresponding to different internal microenvironments. Bacteria were excited at 740 nm and 900 nm and TPEF and FLIM signals were recorded with fitting bandpass filters, respectively. Different species (Escherichia coli K12, Pseudomonas fluorescens and Staphylococcus aureus) and different growth phases were examined to identify characteristic signal combinations. The two image channels and both wavelengths are compared and plotted against each other. The results show that the bacteria can already be classified by their autofluorescence signals. The growth phase seems to have less influence than the species. Based on this data, a classification is made to uniquely identify them. For this purpose, the database will next be expanded to include additional species and the classification will be automated.

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Biofabrication of medical implants

Stiesch

2016

BioNanoMaterials

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Nonlinear laser scanning microscopy of oral multispecies-biofilms: fixative induced fluorescence as a fast and economical in vitro screening method

Cited by 3 publications

References 14 publications

An oral multispecies biofilm model for high content screening applications

An oral multispecies biofilm model for high content screening applications

Multimodal imaging of bacterial species for label-free classification

Biofabrication of medical implants

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