Microscopy

• US 20040100636A1
• Filed: 06/20/2003
• Published: 05/27/2004
• Est. Priority Date: 05/30/2000
• Status: Active Grant

• Current Assignee: University of Nottingham
• Original Assignee: University of Nottingham
• Inventors: Somekh, Michael Geoffrey, See, Chung Wah
• Granted Patent: US 7,142,308 B2
• Time in Patent Office: Days
• US Class Current: 356/497
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G02B 21/002 Scanning microscopes scanning near field optical microscopes G01Q60/18

Abstract

A method and apparatus (1) for analysing a sample (1) are provided. The method comprises the steps of: (i) irradiating the sample with a beam of radiation (4) in which at least some of the radiation is incident on the sample at an angle or angles which results in the excitation of surface waves, (ii) exciting surface waves at a surface of the sample, which surface waves are confined to an area having a principal dimension which is comparable to the wavelength of the surface waves, (iii) detecting radiation which includes radiation produced by the surface waves, and (iv) analyzing the detected radiation to obtain information about the sample. The surface waves may be excited over an annulus of the sample surface, achieved by placing the sample at a position off the focal plane of the apparatus. The surface waves may be surface plasmons. The detected radiation may include radiation irradiated from the sample surface which has excited surface plasmons therein and radiation irradiated from the sample surface which has not excited any surface plasmons therein. The signal generated by the combination of these radiations may be combined with a reference beam of radiation by allowing them to interfere with each other, and the overall signal analysed to obtain information about the sample.

Claims

1. A method of analysing a sample using a microscopy apparatus comprising the steps of:

(i) irradiating the sample with a beam of radiation in which at least some of the radiation is incident on the sample at an angle or angles which results in the excitation of surface waves,

(ii) exciting surface waves at a surface of the sample;

(iii) confining the surface waves to an area having a principal dimension which is comparable to the wavelength of the surface waves by allowing the surface waves to come to a focus;

(iv) detecting radiation which includes radiation produced by the surface waves, and

(v) analysing the detected radiation to obtain information about the sample.

2. A method according to claim 1 characterised in that the surface waves are excited over a substantially circular or elliptical area of the sample surface, and are allowed to come to a focus at substantially the centre of the area.

3. A method according to claim 2 characterised in that excitation over a substantially circular or elliptical area is achieved by placing the sample at the focal plane of the microscopy apparatus.

4. A method according to claim 1 characterised in that the surface waves are excited over an annulus of the sample surface, and are allowed to come to a focus at substantially the centre of the annulus.

5. A method according to claim 4 characterised in that excitation over an annulus is achieved by placing the sample at a position off the focal plane of the microscopy apparatus.

6. A method according to claim 5 characterised in that the position of the sample off the focal plane of the microscopy apparatus is varied to determine the optimum defocus at which the sample should be positioned for analysis.

7. A method according to any preceding claims characterised by using a microscopy apparatus which comprises a means to excite surface waves which means comprise a fluid immersion objective lens.

8. A method according to any preceding claim characterised in that the method is a far field method, i.e. the excitation of the surface waves and the detection of the radiation generated by the surface waves is achieved from the far field.

9. A method according to any preceding claim characterised in that the sample is scanned using scanning means to provide information about a number of points thereof.

10. A method according to any of claims 1 to 8 characterised in that the method comprises using a microscopy apparatus having a widefield configuration.

11. A method according to claim 10 characterised in that the method comprises passing a beam of radiation through a diffuser, and using the resultant beam to irradiate the sample and a reference mirror each with a speckle pattern, causing the radiation from these to be interfered, and detecting the interference signal.

12. A method according to claim 11 characterised in that the method comprises changing the interference conditions between different parts of the radiation irradiating the sample, and critically (as opposed to Kö

hler) illuminating the sample and coherently adding radiations generated by the excitation of surface waves.

13. A method according to any preceding claim characterised in that the sample comprises a conducting material with one or more dielectric materials deposited on or attached to a surface of the conducting material.

14. A method according to any preceding claim characterised in that two or more of the surface waves together produce fluorescenc in the sample.

15. A method according to claim 14 characterised in that the detected radiation include fluorescent radiation from the sample.

16. A method according to claim 13 or claim 14 characterised in that when the sample comprises a metal layer with one or more dielectric materials deposited on or attached to a surface thereof, the metal is chosen to be at least substantially opaque to the radiation used to irradiate the sample and substantially transparent to the fluorescent radiation from the sample.

17. A method according to any of claims 14 to 16 characterised in that the sample has one or more fluorophores added to it and the detected fluorescent radiation is affected by the presence of the fluorophores, and will provide information about the sample.

18. A method according to any of claims 14 to 17 characterised by comprising at least partially blocking radiation reflected from the sample.

19. A method according to any preceding claim characterised in that the method comprises exciting harmonic surface waves.

20. A method according to claim 19 characterised in that the detected radiation comprises radiation produced by the harmonic surface waves.

21. A method according to claim 20 characterised in that the radiation produced by the harmonic surface waves is combined with a reference beam of radiation.

22. A method according to claim 21 characterised in that the radiations are combined by causing them to interfere with each other, using an apparatus provided with or configured as an interferometer.

23. A method according to any preceding claim characterised in that the surface waves which are excited are surface plasmons.

24. A method according to claim 23 characterised in that the detected radiation includes radiation irradiated from the sample surface which has excited surface plasmons therein and radiation irradiated from the sample surface which has not excited any surface plasmons therein.

25. A method according to claim 24 characterised in that one or more characteristics of these radiation are detected and analysed, the one or more characteristics comprising the phase and/or amplitude of the radiation.

26. A method according to claim 24 or claim 25 characterised in that the radiation irradiated from the sample surface which has excited surface plasmons therein and the radiation irradiated from the sample surface which has not excited any surface plasmons therein are combined with a reference beam of radiation by allowing them to interfere with each other, and the overall interference signal generated by the interference of the radiations and the reference beam of radiation analysed to obtain information about the sample.

27. A method according to claim 26 characterised by using a microscopy apparatus which comprises a first sample irradiation arm and a second reference beam arm.

28. A microscopy apparatus which is adapted to carry out the method according to the any of claims 1 to 27.

29. An apparatus according to claim 28 characterised in that it is a far field apparatus, i.e. the excitation of the surface waves and the detection of radiation generated by the surface waves is achieved from the far field.

30. An apparatus according to claim 28 or claim 29 characterised in that the apparatus comprises a radiation source, which produces the beam of radiation used to irradiate the sample.

31. An apparatus according to claim 30 characterised in that the radiation source comprises a laser.

32. An apparatus according to claim 31 characterised iii that the laser is a continuous wave laser.

33. An apparatus according to claim 31 characterised in that the laser is a pulsed laser.

34. An apparatus according to any of claims 28 to 33 characterised in that the apparatus comprises a fluid immersion objective lens through which the sample is irradiated by the beam of radiation.

35. An apparatus according to any of claims 28 to 34 characterised in that the apparatus comprises one or more radiation photodetectors.

36. An apparatus according to any of claims 28 to 35 characterised in that the apparatus comprises analysing means.

37. An apparatus according to any of claims 28 to 36 characterised in that the apparatus comprises scanning means which allow the sample to be scanned to provide information about a number of points thereof.

38. An apparatus according to any of claims 28 to 36 characterised in that the apparatus has a widefield configuration which involves irradiating the sample over a range of angles, covering an extended area of the sample and obtaining information about a number of points of the sample without the need to employ any scanning technique.

39. An apparatus according to claim 38 characterised in that the widefield microscopy apparatus comprises a diffuser, which is used to diffract the radiation used to irradiate the sample over a range of angles.

40. An apparatus according to claim 38 or claim 39 characterised in that the widefield microscopy apparatus comprise interference means, which are used to combine radiation from the sample and a reference beam of radiation.

41. An apparatus according to claim 38 or claim 39 characterised in that the widefield microscopy apparatus comprises means to change the interference conditions between different parts of the radiation irradiating the sample.

42. An apparatus according to any of claims 28 to 41 characterised in that the apparatus comprises sample moving means, for varying the position of the sample off the focal plane of the microscopy apparatus, i.e. vary the defocus z of the sample.

43. An apparatus according to any of claims 28 to 42 characterised in that the apparatus comprises a polariser.

44. An apparatus according to any of claims 28 to 43 characterised in that the apparatus is provided with an interferometer.