Plasmonic imaging and detection of single DNA molecules
Method and system to remove background noise with a differential approach in optical imaging is disclosed. The differential approach moves the sample position laterally over a small distance, and a differential image is generated from the images recorded before and after the lateral translation. This approach can significantly improve the image quality of objects, including single DNA molecules, for label-free optical imaging techniques, such as surface plasmon resonance imaging. Disclosed imaging technique provides high-resolution genome-wide restriction maps of single DNA molecules.
1. A method for label-free optical detection and imaging of a single molecule, the method comprising the steps of:
(a) illuminating a sample attached to a substrate with light,
(b) conditioning the light at the sample,
(c) recording a first image of a sample, acquired in die conditioned light, with an optical detector at a first mutual orientation between light-collecting optics and the substrate;
(d) changing a location of at least one of the light-collecting optics and the substrate from the first location to a second location; and
(e) forming a differential image based on the first image and optical data received by the detector after said changing to obtain an image of said single molecule in the sample,
wherein any of said illuminating, conditioning, recording, changing the location, and forming is carried out without fluorescent labelling of said single molecule.
2. A method according to claim 1 , wherein said changing a location includes spatially translating the substrate between first and second positions with respect to the light-collecting optics.
3. A method according to claim 2 , wherein said spatially translating includes spatially translating the substrate over a distance on the order of wavelength of light.
4. A method according to claim 1 , wherein said illuminating a sample includes illuminating said molecule on a metal surface of the substrate and the metal surface with light, and wherein said conditioning includes scattering light at the sample.
5. A method according to claim 1 , wherein said illuminating includes illuminating a metal surface containing one or more of gold, silver, copper, aluminum, magnesium, platinum, palladium, cobalt, chromium, and nickel, and further comprising creating a surface plasmon wave propagating along said metal surface in a first direction.
6. A method according to claim 1 , wherein said illuminating includes illuminating the substrate, which comprises a dielectric slab coated with a layer of metal, with light at such polarization, wavelength, and incident angle as to excite a surface plasmon wave propagating at the layer of the metal.
7. A method according to claim 1 , wherein said illuminating includes illuminating at least one of a protein, peptide, polypeptide, enzyme, protein-DNA complex, polynucleotide, antibody, DNA, RNA, siRNA, antigen, antigenic epitope, hormone, carbohydrate, lipid, phospholipid, and biotinylated probe.
8. A method according to claim 1 , wherein said forming includes subtracting optical data representing the first image from said optical data received by the detector after said changing.
9. A method according to claim 1 , wherein said forming includes forming the differential image according to:
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wherein
I(x, y)=I S (x, y)+I B (x, y) represents irradiance distribution of an image formed at the first spatial location, I S (x, y) represents irradiance distribution corresponding to an SPR image of the sample, I B (x, y) represents background irradiance distribution formed by causes unrelated to interaction of light with said sample, and I′(x, y)=I S (x, y)+I B (x+Δx, y) represents irradiance distribution of an image formed at the second location, the first and second locations being separated by a distance Δx.
10. A method according to claim 1 , wherein said changing includes repeatedly translating the substrate between the first location and the second location, and said forming includes employing an algorithm to produce an optimal differential image by minimizing background noise.
11. A method according to claim 10 , wherein said employing includes calculating the difference image from images of the sample recorded at the first and second locations for each of repeated translations of the substrate, and using a Fourier filter to selectively remove noise at frequencies different from a frequency at which the substrate is being translated.
12. An apparatus for label-free detecting and imaging of a sample, the apparatus comprising
a substrate on which the sample is attached; a light source illuminating the sample;
a unit configured to spatially translate the substrate between a first and second positions; an optical imaging system disposed to gather and analyze light from said sample to form images at the first and second positions; and
a programmable computer processor configured to produce a differential image representing a difference between said images formed at the first and second positions to obtain an image of a single molecule in the sample,
said apparatus configured to operate and produce said differential image in absence of fluorescent labelling of the sample.
13. An apparatus according to claim 12 , wherein said substrate is coated with a layer of metal and said sample includes said single molecule placed on a metallic surface of said layer, wherein light from said light source is delivered to said substrate such that a surface plasmon wave is excited at the metallic surface, and further including a subsystem configured to collect scattered light associated with said surface plasmon wave.
14. An apparatus according to claim 13 , wherein the metal layer includes at least one of gold, silver, copper, aluminum, magnesium, platinum, palladium, cobalt, chromium, and nickel.
15. An apparatus according to claim 12 , wherein said unit includes at least one of a piezoelectric actuator and an electrical motor.
16. An apparatus according to claim 12 , wherein said optical imaging system comprises a high numerical aperture oil immersion objective.