Scanning analyzer for single molecule detection and methods of use

1. A single molecule analyzer comprising:

(a) an electromagnetic radiation source for providing electromagnetic radiation to a sample container that comprises a sample;

(b) a system for directing the electromagnetic radiation from the electromagnetic radiation source to an interrogation space in the sample;

(c) a translating system for translating the interrogation space through at least a portion of the sample, thereby forming a moveable interrogation space; and

(d) a detector for detecting electromagnetic radiation emitted from a single molecule in the interrogation space if the molecule is present, wherein the detector is operably connected to the interrogation space.

2. The single molecule analyzer of claim 1 wherein the translating system can translate the interrogation space in one or more of a linear path and a non-linear path.

3. The single molecule analyzer of claim 2 wherein the non-linear path comprises a substantially circular path.

4. The single molecule analyzer of claim 2 wherein the non-linear path comprises a helical path.

5. The single molecule analyzer of claim 2 wherein the non-linear path comprises a raster pattern.

6. The single molecule analyzer of claim 1 further comprising a container with a surface for containing and confining at least one sample on the surface.

7. The single molecule analyzer of claim 1 wherein the sample container is a plate.

8. The plate of claim 7 wherein the plate is a microtiter plate.

9. The single molecule analyzer of claim 1 wherein the interrogation space is of a volume between about 15 μm 3 and about 11000 μm 3 .

10. The single molecule analyzer of claim 1 wherein the interrogation space is of a volume between about 200 μm 3 and about 3000 μm 3 .

11. The single molecule analyzer of claim 1 wherein the interrogation space is of a volume between about 500 μm 3 and about 600 μm 3 .

12. The single molecule analyzer of claim 6 wherein the molecule is attached to the surface of the container.

13. The single molecule analyzer of claim 12 wherein a noncovalent bond attaches the molecule to the surface of the container.

14. The single molecule analyzer of claim 13 wherein the noncovalent bonds are formed between the molecule and one or more antibodies covalently or non-covalently bound to the surface of the container.

15. The single molecule analyzer of claim 1 further comprising a microscope objective wherein a depth of field of the microscope objective and a diameter of an aperture imaged to the microscope objective together define the interrogation space.

16. The single molecule analyzer of claim 1 further comprising a microscope objective wherein a depth of field of the microscope objective and a lateral extent of an electromagnetic radiation beam together define the interrogation space.

17. The single molecule analyzer of claim 1 wherein the translating system is constructed and arranged to translate the interrogation space through the portion of sample more than one time.

18. The single molecule analyzer of claim 17 wherein the translating system is constructed and arranged to translate through a same portion of sample a first time and a second time at a sufficiently slow speed as to allow a molecule of interest, if present, detected the first time the interrogation space is translated through the portion of sample to substantially diffuse out of the portion of sample after the first time the portion of sample is interrogated by the interrogation space and to further allow a subsequent molecule of interest, if present, to substantially diffuse into the portion of sample the second time the portion of sample is interrogated by the interrogation space.

19. The analyzer of claim 18 wherein the translating system is constructed and arranged to translate the interrogation space in a substantially circular pattern, wherein the system is capable of translating the interrogation space at a speed of between about 100 and about 1000 RPM.

20. The single molecule analyzer of claim 17 wherein the translating system is constructed and arranged to translate the interrogation space such that the detection spot returns to the portion of sample after sufficient time has passed so that the molecule detected in the first pass can diffuse out of the portion, and other molecules can diffuse into the portion.

21. The single molecule analyzer of claim 1 wherein the analyzer is adapted and configured to sequentially detect the presence or absence of a single molecule of a particular type in a first sample, and detect the presence or absence of a single molecule of the type in a second sample, wherein there is no carryover between the first and the second sample.

22. A method for detecting the presence or absence of a single molecule in a sample comprising:

(a) directing electromagnetic radiation from an electromagnetic radiation source to an interrogation space in the sample;

(b) detecting the presence or absence of a first single molecule in the interrogation space located at a first position in the sample;

(c) translating the interrogation space through the sample to a subsequent position in the sample;

(d) detecting the presence or absence of a subsequent single molecule in the subsequent position in the sample; and

(e) repeating steps (c) and (d) as required to detect the presence or absence of a single molecule in more than one position of the sample.

23. The method of claim 22 wherein the interrogation space is of a volume between about 15 μm 3 and about 11000 μm 3 .

24. The method of claim 22 wherein the interrogation space is of a volume between about 200 μm 3 and about 3000 μm 3 .

25. The method of claim 22 wherein the interrogation space is of a volume between about 500 μm 3 and about 600 μm 3 .

26. The method of claim 22 wherein the interrogation space is translated in a non-linear path.

27. The method of claim 26 wherein the non-linear path comprises a substantially circular path.

28. The method of claim 26 wherein the non-linear path comprises a helical path.

29. The method of claim 22 wherein the sample remains substantially stationary relative to the electromagnetic radiation directed at the interrogation space located within the sample.

30. The method of claim 22 wherein the interrogation space is translated through the first position of sample more than one time.

31. The method of claim 30 wherein the interrogation space is translated through the first position of sample a subsequent time at a sufficiently slow speed as to allow a molecule of interest, if present, detected the first time the interrogation space is translated through the position of sample to substantially diffuse out of the position of sample after the first time the position of sample is interrogated by the interrogation space and to further allow a subsequent molecule of interest, if present, to substantially diffuse into the position of sample the second time the position of sample is interrogated by the interrogation space.

32. The method of claim 30 wherein the interrogation space is translated such that the detection spot returns to the first position of sample after sufficient time has passed so that a molecule detected in the first pass can diffuse out of the position, and another molecule can diffuse into the position.

33. The method of claim 22 further comprising the steps of sequentially detecting the presence or absence of a single molecule of a particular type in the sample, then detecting the presence or absence of a single molecule of the same type in a second sample, wherein there is no carryover between the first and the second sample.

34. The method of claim 33 wherein the first sample and the second sample are contained and confined in a non-disposable apparatus.