Antibiotic susceptibility testing via plasmonic imaging and tracking
View Patent ↗A rapid antibiotic susceptibility test (AST) based on the detection and quantification of the movement of single bacterial cells with a plasmonic imaging and tracking (PIT) technology. The PIT-based AST detects changes in the metabolic activity of the bacterial cells long before cell replication, and allows rapid AST for both cultivable and non-cultivable strains. PIT tracks 3D movement with sub-nanometer resolution and millisecond temporal resolution. PIT also allows simultaneous measurement of the binding kinetic constants of antibiotics and bacterial metabolic state after the introduction of antibiotics.
1. A method for antibiotic susceptibility testing using plasmonic imaging for bacterial cells comprising:
providing a plasmonic imaging and tracking (PIT) system including an inverted microscope lens, a light source, a metallic coated slide, a mirror and a detector;
attaching tethering molecules to the metallic coated surface;
populating the metallic coated surface with bacteria;
activating the PIT system;
imaging the bacteria using the PIT system;
tracking a first set of 3D motion values of the bacteria;
adding an antibiotic to the metallic coated surface;
tracking a second set of 3D motion values of the bacteria in the presence of the antibiotic; and
comparing the first and second 3D motion values to determine changes in the 3D motion of the bacteria after addition of the antibiotic.
2. The method of claim 1 wherein attaching tethering molecules comprises attaching tethering molecules with an affinity to a bacterial cell under investigation.
3. The method of claim 2 wherein attaching tethering molecules comprises attaching antibodies.
4. The method of claim 1 wherein populating the metallic coated surface with a bacteria comprises populating the metallic coated surface with a bacteria selected from the group consisting of E. coli and S. aureus.
5. The method of claim 1 wherein attaching tethering molecules comprises attaching tethering molecules including anti- E. coli antibodies.
6. The method of claim 1 wherein attaching tethering molecules comprises attaching tethering molecules selected from the group consisting of cell-adhesion promoting substances, poly-lysine, and agar matrix.
7. The method of claim 1 wherein populating the metallic coated surface with the bacteria comprises tethering the bacteria within a distance of less than five hundred nm from the metallic coated surface.
8. The method of claim 1 wherein tracking a first set of 3D motion values comprises:
extracting an image intensity change from the plasmonic image that is free of noise;
processing a plasmonic image of a bacterial cell where the plasmonic image includes a bright spot with a parabolic shaped tail;
tracking bacteria XY-motion by detecting the bright spot at the vertex of the parabolic shaped tail; and
tracking bacteria Z-motion by detecting substantially perpendicular motion relative to the metallic coated surface.
9. The method of claim 8 wherein tracking the XY-motion comprises using a curve fitting algorithm.
10. The method of claim 9 wherein the curve-fitting algorithm is selected from the group consisting of Gaussian fitting, elliptical fitting, and spatial averaging.
11. The method of claim 8 wherein extracting an image intensity change from the plasmonic image that is free of noise comprises transforming the plasmonic image into K-space using Fourier transforms to produce a two-ring image.
12. A method for antibiotic susceptibility testing using plasmonic imaging for individual bacterial cells comprising:
providing a plasmonic imaging and tracking (PIT) system including an inverted microscope lens, a light source, a metallic coated slide, a mirror and a detector;
attaching tethering molecules to the metallic coated surface;
populating the metallic coated surface with individual bacterial cells;
activating the PIT system;
imaging, using the PIT system, the individual bacterial cells;
tracking a first set of 3D motion values of each of the individual bacterial cells;
adding an antibiotic to the metallic coated surface;
tracking a second set of 3D motion values of each of the individual bacterial cells in the presence of the antibiotic; and
comparing the first and second 3D motion values to determine changes in the 3D motion of each of the individual bacterial cells after addition of the antibiotic.