Apparatus and method for tracking a molecule or particle in three dimensions

• US 20080085550A1
• Filed: 10/06/2006
• Published: 04/10/2008
• Est. Priority Date: 10/06/2006
• Status: Active Grant

• Current Assignee: TRIAD National Security, LLC
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Lessard, Guillaume, Goodwin, Peter M., Werner, James H.
• Granted Patent: US 7,498,551 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1475 using image analysis for extracting features of the particle
• CPC Class Codes: G01N 2015/1027 Determining speed or velocity of a particle
• CPC Class Codes: G01N 2015/1075 Determining speed or velocity of a particle
• CPC Class Codes: G01N 2021/1785 Three dimensional
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/4795 spatially resolved investigating of object in scattering medium in vivo A61B
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

An apparatus and method were used to track the movement of fluorescent particles in three dimensions. Control software was used with the apparatus to implement a tracking algorithm for tracking the motion of the individual particles in glycerol/water mixtures. Monte Carlo simulations suggest that the tracking algorithms in combination with the apparatus may be used for tracking the motion of single fluorescent or fluorescently labeled biomolecules in three dimensions.

Claims

1. An apparatus for tracking the position of a molecule or particle in a sample, comprising:

a stage for supporting the sample,

a light source for emitting a light beam capable of inducing a measurable light-based response from a chosen molecule or particle in the sample,

an objective lens for focusing the light beam on the sample to create an excitation volume, said objective lens projecting at least a portion of the light based response from the excitation volume to at least one image plane,

a dichroic element for directing the light beam from the light source to the objective lens,

at least one spatial filter at the at least one image plane for selecting a portion of the light based response and defining an optical probe volume for that portion of the light based response,

means for introducing a controlled relative motion between the optical probe volume and the molecule or particle, and

means for monitoring the light based response from the chosen molecule or particle, wherein said means for monitoring the light based response uses the light based response to control an overlap between the optical robe volume with the particle or molecule being tracked.

2. The apparatus of claim 1, wherein said means for monitoring the light based response comprises a plurality of photon detectors that detect light the light based response from the molecule or particle in combination with a computer system that uses the detected light based response from the photodetectors to determine how said apparatus should be adjusted to maintain the molecule or particle being tracked in the optical probe volume.

3. An apparatus for tracking the position of a molecule or particle in a sample, comprising:

a stage for supporting the sample,

a light source for emitting a light beam capable of inducing a light based response from a chosen molecule or particle in a sample,

an objective lens for focusing the light beam on the chosen molecule or particle in the sample,

means for introducing a controlled lateral displacement between said light source and said stage,

a dichroic element for directing the light beam from said light source to said objective lens,

means for creating at least a first image plane and a second image plane, the first image plane including spatial information from a first portion of the sample, the second image plane including spatial information from a second portion of the sample, the first portion and second portion defining an optical probe volume,

a first optical spatial filter that sends light from the first image plane to a first photon detector and a second optical spatial filter that sends light from the second image plane to a second photon detector,

a first photon detector for receiving light from the first image plane and a second photon detector for receiving light from the second image plane, and

means for analyzing light from said first detector and said second detector, wherein said means for analyzing light uses an analysis of light from the first photon detector and the second photon detector to control an overlap between the optical probe volume and the particle or molecule being tracked.

4. The apparatus of claim 3, wherein at least one of said first spatial optical filter and said second spatial optical filter is moveable.

5. The apparatus of claim 3, wherein said means for creating at least a first image plane and a second image plane comprises a beamsplitter or a dichroic filter.

6. The apparatus of claim 3, wherein said means for reading the photon counts and using the photon counts to control the overlap between the optical probe volume and the molecule or particle being tracked comprises a plurality of photodetectors that detect the light based response from the molecule or particle in combination with a computer system that uses the detected light based response from the photodetectors to determine how said apparatus should be adjusted to maintain the molecule or particle being tracked in the optical probe volume.

7. The apparatus of claim 3, further comprising a tube lens for focusing a portion of the light emission onto the first image plane, and for focusing another portion of the light emission onto the second image plane.

8. An apparatus for tracking the position of a molecule or particle in a sample, comprising:

a light source for emitting a light beam capable of inducing fluorescence from a chosen molecule or particle in a sample,

an objective lens for focusing the light beam on the chosen molecule or particle in the sample,

a stage for supporting the sample,

a dichroic element for directing the light beam from the light source to the objective lens,

means for creating a first image plane and a second image plane, the first image plane including spatial information from a first portion of the sample, the second image plane being nearly conjugate with the first image plane and including spatial information from a second portion of the sample, the first portion and second portion defining an optical probe volume,

a first optical fiber that sends light from the first image plane to a first photon detector, the first optical fiber having a cross-section with a center,

a second optical fiber that sends light from the first image plane to a second photon detector, the second optical fiber having a cross-section with a center, wherein a line connecting the center of the first optical fiber and the center of the second optical fiber is parallel to the x-axis,

a third optical fiber that sends light from the second image plane to a third detector, the third optical fiber having a cross-section with a center,

a fourth optical fiber that sends light from the second image plane to a fourth detector, the fourth optical fiber having a cross-section with a center, wherein a line connecting the center of third optical fiber and the center of the fourth optical fiber is parallel to the y axis, and

means for reading photon counts from fluorescence detected by said first detector and said second detector and said third detector and said fourth detector, wherein said means for reading photon counts uses the photon counts to control the overlap between the optical probe volume and the particle or molecule being tracked.

9. The apparatus of claim 8, wherein at least one of the first optical spatial filter, the second optical spatial filter, the third optical spatial filter, and the fourth optical spatial filter comprises a circular optical fiber.

10. The apparatus of claim 8, wherein said stage is moveable in order to adjust of the position of the particle or molecule being tracked.

11. The apparatus of claim 8, wherein at least one of the first optical spatial filter, the second optical spatial filter, the third optical spatial filter, and the fourth optical spatial filter is movable.

12. The apparatus of claim 8, wherein said means for creating the first image plane and the second image plane comprises an intensity beamsplitter that splits the fluorescent light emitted from the chosen molecule or particle.

13. The apparatus of claim 8, wherein said means for creating the first image plane and the second image plane comprises a polarization beamsplitter or a dichroic filter.

14. The apparatus of claim 8, wherein said means for reading photon counts from fluorescence detected by said first detector and said second detector and said third detector and said fourth detector and using the photon counts to control the position of said moveable sample stage comprises a computer system and computer readable software that the computer uses in combination with the photon counts to control the position of the piezo stage so that the chosen particle or molecule remains substantially in the center of an optical volume.

15. The apparatus of claim 8, wherein said means for reading photon counts from fluorescence detected by said first detector and said second detector and said third detector and said fourth detector, said means using the photon counts to control the position of said moveable sample stage comprises a field programmable gate array (FPGA).

16. The apparatus of claim 8, further comprising a tube lens for focusing light collected from the objective lens alternately onto the first image plane and onto the second image plane.

17. A method for tracking the position of a particle or molecule in a sample, comprising:

exposing a sample to light to induce fluorescence from a chosen particle or molecule in the sample, the sample comprising a plurality of z-slices that define an optical probe volume;

analyzing the fluorescence with a first image plane that includes spatial information from a first z-slice from the plurality of z-slices, and with a second image plane that is nearly conjugate with the first image plane;

the second image plane including spatial information from a second z-slice from the plurality of z-slices; and

using the spatial information from the first image plane and the second image plane to control the overlap of the optical probe volume and the chosen particle or molecule being tracked.

18. A method for tracking the position of a particle or molecule in a sample, comprising:

exposing a sample to light for a chosen period of time to induce light emission from a chosen particle or molecule in the sample;

detecting the light emission using a plurality of detectors; and

using the intensity of the light emission to evaluate the distance between the center of an optical probe volume and the chosen molecule or particle being tracked.

19. The method of claim 18, further comprising using the intensity of the light emission to control an overlap of the optical probe volume with the particle or molecule being tracked.

20. A method for tracking a molecule in three dimensions, comprising:

exposing a sample to light for a chosen period of time to induce light emission from a chosen particle or molecule in the sample,

detecting the light emission using a plurality of detectors, each of said plurality recording the light emission at a different location near the sample,

determining which of the plurality of detectors detected the highest light emission during the chosen period of time,

adjusting the position of the sample away from the detector that detected the highest light emission and toward the other detectors of the plurality of detectors.