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Progress of Rapid Optical Detection Methods for Foodborne Pathogens
Bosoon Park (Lead Scientist), Seung-Chul Yoon, Kurt Lawrence, Gary Gamble
USDA, ARSU.S. National Poultry Research Center
Quality & Safety Assessment Research UnitAthens, Georgia
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Outline
Early and rapid detection of Salmonella with hyperspectral microscope imaging at a cellular level
High-throughput foodborne pathogen detection with surface plasmon resonance imaging
High-speed hyperspectral imaging for foreign material separation
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Hyperspectral Microscope Imaging (HMI)
AOTF Hyperspectral Imaging System
• Acousto-optical tunable filter (AOTF) hyperspectral imaging system
• High-speed, high-throughput, random access
• Solid-state optical filter with adjustable optical bandpass
• Wavelength & bandwidth changed less than 100 ms
• Tuning range: 450-800 nm• Bandwidth: 1.5 nm (@450
nm), 3 nm (@800 nm)• Output: linear polarized• EMCCD attached
EMCCD Camera
AOTF
Microscope AOTF Controller
Stage
Light Source
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Hyperspectral Microscope Imaging (HMI)
Hyperspectral Images from Salmonella Enteritidis19 Selected from 89 Contiguous Images with 450-800nm
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Hyperspectral Microscope Imaging (HMI)
Hyperspectral Images of Salmonella under Metal-Halide and Tungsten-Halogen Lights
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Hyperspectral Microscope Imaging (HMI)
Comparison of Spectra from Salmonella under Different Light Sources (Halide vs. Halogen)
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Hyperspectral Microscope Imaging (HMI)
3-D Scattering Intensity Distribution of Bacterial Cells
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Hyperspectral Microscope Imaging (HMI)
Comparison of Spectra from Gram-positive and Gram-negative Bacteria
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Hyperspectral Microscope Imaging (HMI)
Score Plots of Gram-positive and negative Bacteria
Salmonella (black) vs. Staphylococcus (red)
Accuracy: 96.7% (PLS-DA)
Three Gram-negatives
E. coli, S. Kentucky, Citrobacter
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Hyperspectral Microscope Imaging (HMI)
Classification Accuracy of Different ModelsGram-negative vs. Gram-positive
• Total number of data: 147,540
• Calibration/Validation (45%): 65,663
• Testing (55%): 81,877
Notes: G+ includes Staphylococcus (aureus, haemolyticus, hyicus, sciuri, and simulans) and G-includes Salmonella (Enteritidis, Typhimurium, Kentucky, Heidelberg, Infantis)
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Hyperspectral Microscope Imaging (HMI)
Classification Accuracy of Different ModelsSalmonella 5 Serotypes
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Hyperspectral Microscope Imaging (HMI)
Procedure of Early and Rapid Detection
Sample Preparation
24 hrs Incubate Salmonella
8 hrs Pick Colonies
Suspend Colonies
Prepare Glass Slide
10 min Air Dry
Cover Slip
Data Analysis
45 sec
Acquire Hyperspectral Image (TIFF)
Convert to Hypercube ROIs
Selected in ENVI
Data Randomized
Classification Modelingk-NN, LDA,
QDA, SVM, PLS-DA
Select Optimum
Model
• Data converted from 3-D hypercube
• Incubation time can be reduced from 24 hrs to 8 hrs
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Spectral Comparison with Different Growing Time
S. Enteritidis
Wavelength (n.m.)
500 600 700 800
Inte
nsity
(a.u
.)
0
1000
2000
3000
4000
5000
6000
7000
8000
8 Hr
10 Hr
12 Hr
24 Hr
Serotype 8:24 hrs 10:24 12:24
S. Enteritidis 0.9944* 0.9959* 0.9869*
S. Heidelberg 0.9946* 0.9975* 0.9947*
S. Infantis N/A 0.9967* 0.9971*
S. Kentucky 0.9953* 0.9926* 0.9957*
S. Typhimurium 0.9990* 0.9950* 0.9990*
*Pearson Correlations for each serotype, comparing 8, 10, 12 to 24h
• All serotypes showed similar spectral patterns with peaks at same locations and intensities varied with times
• The table shows the spectral features between 8, 10 and 12 hrs to 24 hrs are similar
• Time does not appear to affect on spectral patterns, apart from increasing intensities
• Spectral signatures were not affected by growing time that means once cells are grown, their spectral signatures are similar in same serotype
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Classification of Salmonella Serotypes with Different Incubation Time (8 hrs vs. 24 hrs)
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
-6
-4
-2
0
24
6
-1.0-0.8-0.6-0.4-0.20.00.20.40.60.8
PC
3
PC 1
PC 2
8 hrs PCA Score Plot
S. Enteritidis
S. Heidelberg
S. Kentucky
S. Typhimurium
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
-8-6
-4-2
02
46
810
-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.6
PC
3
PC 1
PC 2
24 hrs PCA Score Plot
S. Enteritidis
S. Heidelberg
S. Infantis
S. Kentucky
S. Typhimurium
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Surface Plasmon Resonance Imaging (SPRi)
High-throughput Foodborne Pathogen Detection with SPRi
Chen and Park, Analy. & Bioanaly. Chem. DOI 10.1007/s00216-017-0810-z (2017)
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Surface Plasmon Resonance Imaging (SPRi)
Surface Functionalization of SPRi Sensor Chips
• Automatic arraying of ligand spots: Spot size: 500 µm Average deposition volume: 5 nL/spot Sensor surface area: 0.96 cm x 0.64 cm
• Optimization of ligand immobilization condition was conducted on a single chip. Optimization parameters: pH (4.6, 5.6, and 7.4) Concentration of antibody (10-250 µg/mL)
Microarray pattern design Actual biochip image after ligand immobilization
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Surface Plasmon Resonance Imaging (SPRi)
Optimizing Ligand Immobilization
• Biochip image before SPRi
Ligand spots are visible at concentration ≥ 100 µg/mL
Spot size may exceed 500 µm at high concentrations
• SPR signal intensity after sample injection
Max signal is obtained at pH4.6 and 250 µg/mL
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Surface Plasmon Resonance Imaging (SPRi)
SPR Sensorgram and Analysis
• As anti-Salmonella antibody concentration increases, SPR signal intensity increases• Different serotypes of Salmonella have different binding kinetics at the same cell
concentration and yielded different signals• No cross-reaction with negative control species
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Surface Plasmon Resonance Imaging (SPRi)
SPRi Image Analysis• Flow cell images at the beginning of each
injection is set as reference images.
• Difference images are generated by subtracting real-time flow cell images during each injection by the reference image.
Biochip image
Bright spots for Salmonellaspecies
No response in negative control species
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Surface Plasmon Resonance Imaging (SPRi)
Regeneration of Sensor Surface
• After each injection, the sensor surface is regenerated by injecting acidic or alkaline buffers to disrupt antigen-antibody bonding.
• After regeneration, the sensor surface is ready for new sample injection.
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Two-pathogen screening (S. Typhimurium and O157)
Multiplex and Label-Free Screening of Foodborne Pathogens Using Surface Plasmon Resonance Imaging (Poster)
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High-Speed Hyperspectral Imaging
• One system for real-time disease and/or fecal detection system
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High-Speed Hyperspectral Imaging 2
• Universal hyperspectral machine vision guided-real-time foreign material detection system
Hyperspectral Imaging System Robotic Vacuum Line
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High-Speed Hyperspectral Imaging 3
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Future Work
• Efficient sampling methods and protocols from food matrices for hyperspectral microscope imaging (HMI) scan.
• Improve limit of detection (LOD) for surface plasma resonance imaging (SPRi).
• Applying robotics for real-time separation of foreign materials during poultry processing.
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Acknowledgements
• Jing Chen (Postdoc.)
• Matthew Eady (UGA graduate student)
• Nasreen Bano (Technician)
• Arthur Hinton, Jr. (Poultry Microbiological Safety & Processing RU)