Development of a multispectral light‐scatter sensor for bacterial colonies

Abstract

We report a multispectral elastic-light-scatter instrument that can simultaneously detect three-wavelength scatter patterns and associated optical densities from individual bacterial colonies, overcoming the limits of the single-wavelength predecessor. Absorption measurements on liquid bacterial samples revealed that the spectroscopic information can indeed contribute to sample differentiability. New optical components, including a pellicle beam splitter and an optical cage system, were utilized for robust acquisition of multispectral images. Four different genera and seven shiga toxin producing E. coli serovars were analyzed; the acquired images showed differences in scattering characteristics among the tested organisms. In addition, colony-based spectral optical-density information was also collected. The optical model, which was developed using diffraction theory, correctly predicted wavelength-related differences in scatter patterns, and was matched with the experimental results. Scatter-pattern classification was performed using pseudo-Zernike (GPZ) polynomials/moments by combining the features collected at all three wavelengths and selecting the best features via a random-forest method. The data demonstrate that the selected features provide better classification rates than the same number of features from any single wavelength. Three wavelength-merged scatter pattern from E. coli.

Development of the Correction Algorithm to Limit the Deformation of Bacterial Colonies Diffraction Patterns Caused by Misalignment and Its Impact on the Bacteria Identification in the Proposed Optical Biosensor

“…The proposed approach enables the identification of bacterial colonies with accuracy higher than 98% [ 5 ]. Similar results were also achieved by alternative methods based on forward scattering light patterns [ 1 , 2 , 3 , 17 , 18 , 19 ]. However, it is essential to identify and, in the end, eliminate any non-specific factors potentially having an impact on the accuracy of the identification.…”

“…Therefore, it was important to take the misalignment into account for all alternative configurations of optical biosensors based on diffraction or forward-light scattering patterns [ 6 , 7 , 17 , 19 ]. It was particularly critical, when the features extraction and classification were carried out applying Chebyshev, Legendre, or Zernike moments [ 6 , 17 , 19 ], as they depend on the object’s symmetry [ 27 , 28 ]. As it was demonstrated above, any deviation of the optical signature’s symmetry caused by the misalignments, affected the classification accuracy.…”

“…The impact of the misalignment was not analyzed for the alternative identification methods [ 6 , 7 , 17 , 19 ] and no attempts were made to quantitatively characterize its influence on classification accuracy. Some of these methods are based on the Zernike moments/polynomials [ 6 , 17 , 19 ], which are unique tools to capture information on circular patterns for feature extraction and classification. In this case, circular scattering patterns were downsized, centered, and the basic Zernike functions (being the complex radial polynomials exhibiting non-zero values inside of a united circle defined in polar coordinates) were evaluated.…”

“…The misalignment can be introduced by the sample scanning module and is associated with the minimal achievable movement increment of the used motorized X–Y translational stages and applied scanning and system centering protocols. It should also be taken into consideration with the other alternative methods based on the forward scattering patterns of bacterial colonies, particularly when the features extraction and classification are based on other approaches, such as the use of Zernike (GPZ) polynomials/moments [ 6 , 17 , 25 ], which are highly sensitive for any variations of optical signatures symmetry. According to our knowledge, the influence of this system-dependent factor on deformation of diffraction patterns has never been reported before.…”

Generalized spectral light scatter models of diverse bacterial colony morphologies

“…However, the single‐wavelength model has a limitation in classifying organisms at lower hierarchical levels . For several years, researchers have worked on developing innovative BARDOT approaches, resulting in the introduction of multispectral and reflective BARDOT instruments .…”