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Hyperspectral fluorescence in-situ hybridization (FISH) imaging for simultaneous detection and quantification of foodborne bacterial pathogens

Seung-Chul Yoon 1 ; Brian Oakley 2 ; Bosoon Park 1 ; Kurt Lawrence 1 ; Johnna Garrish 1 ; Eirc Line 1
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Introduction:
Simultaneous detection and identification of multiple foodborne pathogens is important for numerous food safety applications. Hyperspectral fluorescence in-situ hybridization (FISH) imaging is a molecular technique that conducts multiplex FISH imaging with hyperspectral fluorescence microscopy for simultaneous detection and enumeration of whole bacterial cells. The detection principle of standard FISH is based on binding fluorescent oligonucleotide probes to regions of ribosome RNAs (rRNA), thus providing almost any level of taxonomic resolution. A limiting factor of the standard FISH method is being able to detect only a few dyes simultaneously due to the spectral crosstalk of adjacent fluorescent emission spectra which are highly overlapped. This paper will present the research on hyperspectral microscope imaging with spectral unmixing to simultaneously and differentially detect multiple bacterial cells specifically labelled with FISH probes.

Materials and Methods:
Probe design and bacterial cultures:
A total of seven FISH probes were designed and optimized for bacterial cultures associated with poultry and poultry processing environments, including Campylobacter sp., Salmonella sp., Clostridium perfringens, Arcobacter sp., Listeria sp., Staphylococcus aureus, and Pseudomonas sp. Some of the 16S rRNA-targeted oligonucleotide probes were selected from probeBase and commercially synthesized with specific fluorophore modifications by Integrated DNA Technologies or Eurofins MWG/Operon. Others were designed de novo with the ARB software package using a comprehensive set of rRNA sequences obtained from the Silva reference database.

FISH protocol:
Paraformaldehyde fixed cells were transferred onto gelatin coated slides and incubated at 46°C in a moisture chamber with a standardized concentration of 25% formamide (FA) buffer containing an oligonucleotide probe. After a short washing in corresponding FA wash buffer, the cells were mounted with antifading reagent for quick microscopic visualization and color image acquisition using an Olympus BX-60 fluorescent microscope.

Hyperspectral microscope imaging (HMI):
Hyperspectral microscope images of fluorescent bacterial cells were acquired with two different hyperspectral microscope systems, specifically in the 450-800 nm spectral range using an AOTF-based widefield hyperspectral microscope (Nikon-ChromoDynamics Hsi-400) and in the 400-800 nm spectral range with a spectral confocal laser scanning microscope (Olympus FluoView FV1200). The parameters for HMI, such as exposure time, detector gain, and wavelength ranges for spectral scanning, bandwidths, and intensity of lasers for confocal system were optimized to increase the signal intensity and the signal-to-noise ratio. To build and process hyperspectral images, we constructed a high-throughput data analysis pipeline with Matlab scripts. A spectral unmixing method was applied for detection and differentiation of the seven types of bacterial cells.

Results and Discussion:
Increasing the fluorophore concentration to 300-500 ng/µl with extension of the hybridization time to five hours helped to increase the target accessibility of the FISH probes. Preliminary study results done with pure cultures showed the unambiguous differentiation of all seven bacteria when combined with a linear unmixing technique of acquired hyperspectral images. An automatic cell segmentation algorithm was developed as a part of a future system for automation of scanning and analysis. The sensitivity and specificity of the developed hyperspectral FISH imaging still need to be evaluated with mixed cultures and a food matrix.