Autofluorescence Detection Method for Dental Plaque Bacteria Detection and Classification: Example of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Streptococcus mutans

Abstract

The use of fluorescence spectroscopy for plaque detection is a fast and effective way to monitor oral health. At present, there is no uniform specification for the design of the excitation light source of related products for generating fluorescence. To carry out experiments on dental plaque, the fluorescence spectra of three different bacterial species (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Streptococcus mutans) were measured by hyperspectral imaging microscopy (HIM). Three critical issues were found in the experiments. One issue was the unwanted spectrum generated from a mercury line source; two four-order low-pass filters were evaluated for eliminating the unwanted spectrum and meet the experimental requirements. The second issue was the red fluorescence generated from the microscope slide made of borosilicate glass; this could affect the observation of the red fluorescence from the bacteria; quartz microscope slides were found to reduce the fluorescence intensity by about 2 dB compared with the borosilicate slide. The third issue of photobleaching in the fluorescence of the Porphyromonas gingivalis was studied. This study proposes a method of classifying three bacteria based on the spectral intensity ratios (510/635 and 500/635 nm) under the 405 nm excitation light was proposed in this study. The sensitivity and specificity of the classification were approximately 99% and 99%, respectively.

Development of a Visualisation Approach for Analysing Incipient and Clinically Unrecorded Enamel Fissure Caries Using Laser-Induced Contrast Imaging, MicroRaman Spectroscopy and Biomimetic Composites: A Pilot Study

“…There is also a specific gradation of colours in the images that allows the visualisation of the bacterial invasion. This agrees with the spectral analysis data and known works that have demonstrated the different contributions of porphyrins and microorganisms producing these substances at the different stages of caries [ 12 , 24 , 25 , 56 , 57 ]. At the same time, for the initial stages of caries (ICDAS 1–2), the use of the visualisation mode without a filter application, that is, observation of the reflected signal, is less informative ( Figure 3 , line II).…”

“…Complex analysis of the images in Figure 3 demonstrates that the fluorescence intensity separated by the notch filter is considerably higher in the regions surrounding the destroyed dental tissue. It is well known that under the laser-induced fluorescence at 532 nm, the emission bands of porphyrins (associated with the bacteria resulting in caries) can be stimulated [ 12 , 24 , 25 , 56 , 57 ]. In this case, the intense fluorescence detected in the red spectral range [ 12 , 57 , 58 ] indicates not only an increase in the microorganism content in the area of contrast but also implies possible disorganisation processes in the dental tissue, i.e., the changes are already occurring in the organic components of enamel and dentin in the investigated teeth ( Figure 3 , line III).…”

“…The information obtained from these methods makes it possible to describe not only the crystal and chemical characteristics of the apatite that comprises the dental tissue but also the chemical transformations in the organic component of teeth [ 21 ]. An important feature of these spectroscopic methods is their ability to determine the bacterial status of the detected area and the state of the biofilm [ 22 , 23 , 24 ].…”

“…The main principle is to use low-power laser emissions to scan the dental tissue to detect carious lesions [ 30 ]. The use of different wavelengths can detect differences in the density, structure and chemical composition of the biological tissue, while fluorescence losses are appropriate for assessing the stage and depth of the caries infection [ 12 , 24 ]. Currently, approaches to caries visualisation based on the fluorescence of the intact and carious tissue assist dentists in providing minimally invasive treatment; moreover, they can do so without X-ray analysis [ 28 , 30 , 31 , 32 , 33 ], which can be inefficient, particularly when it is impossible to diagnose an incipient lesion and the destruction of tooth enamel in fissure system [ 26 , 30 , 33 ].…”

Choosing the Probe for Single-Molecule Fluorescence Microscopy

“…Some works mentioned that standard coverslips made of borosilicate glass generate fluorescence in the red region under blue excitation [ 73 , 81 ], green excitation [ 73 , 75 , 82 ] and mercury arc lamp excitation [ 83 ]. This red emission from glass hinders the use of the spectral region usually preferred based on autofluorescence from biological specimens ( Figure 1 B).…”