Prism design for scanning applications and illumination of microscopy sample

• US 20050057798A1
• Filed: 07/16/2004
• Published: 03/17/2005
• Est. Priority Date: 01/16/2002
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Osborne, Mark, Bryant, Jason, Obradovic, Bojan, Swerdlow, Harold
• Granted Patent: US 7,518,764 B2
• Time in Patent Office: Days
• US Class Current: 359/368
• 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
• CPC Class Codes: G02B 26/108 having one or more prisms as scanning elements
• CPC Class Codes: G02B 27/56 Optics using evanescent waves, i.e. inhomogeneous waves
• CPC Class Codes: G02B 5/04 Prisms

Abstract

There is disclosed a prism for use in scanning applications such as total internal reflection microscopy in which the prism is translated relative to an incident light beam. A geometry is disclosed which cancels walk of the beam footprint at the base of the prism. Walk of the beam footprint due to irregularities in a largely planar sample surface located at the prism base are cancelled by coupling movement of the incident light beam to movement of the sample in the field of view of an objective lens, for example as part of an autofocus arrangement.

Claims

1. A scanning apparatus comprising:

—

a light source for generating a light beam; and

an angular prism coupled to a sample at a base interface;

characterised in that the base angle θ

_p of the prism satisfies the equations;

—

$\frac{\left({\cos }^2<br><br>{\mathrm{\theta <br><br>}}_c-{\sin }^2<br><br>{\mathrm{\theta <br><br>}}_p\right)<br><br>\left(\tan <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_i<br><br>\sin <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_p+\cos <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_p\right)=1}{\cos <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_c<br><br>\cos <br><br>\left({\mathrm{\theta <br><br>}}_p\pm <br><br>{\mathrm{\theta <br><br>}}_c\right)}$

and

n_i sinθ

_i=n_p sin (θ

_p−

θ

_c)

wherein θ

_c is the coupling angle required for light incident at base interface of the prism, θ

_i is the incident angle of the light beam on the prism, n_i is the refractive index of the medium at the interface where the light beam enters the prism and n_p is the refractive index of the prism.

2. A scanning apparatus according to claim 1, wherein

θ

_p=θ

_c and θ

_i=0.

3. A total internal reflection microscopy apparatus according to claim 1, including means for detecting interaction of the sample at the base of the prism with an evanescent wave formed by total internal reflection of the light beam at the base of the prism.

4. A total internal reflection microscopy apparatus according to claim 3, wherein

θ

_c≧

sin<sup>−

1</sup>(n_s/n_p)

and wherein n_s is the refractive index of the sample medium.

5. A scanning method comprising the steps of:

—

generating a light beam;

providing an angular prism in the path of the light beam, the prism being coupled to a sample at a base interface; and

moving the prism and sample relative to the light beam;

characterised in that the base angle θ

_p of the prism satisfies the equations;

—

$\frac{\left({\cos }^2<br><br>{\mathrm{\theta <br><br>}}_c-{\sin }^2<br><br>{\mathrm{\theta <br><br>}}_p\right)<br><br>\left(\tan <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_i<br><br>\sin <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_p+\cos <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_p\right)=1}{\cos <br><br>\text{\hspace{1em}<br><br>}<br><br>{\mathrm{\theta <br><br>}}_c<br><br>\cos <br><br>\left({\mathrm{\theta <br><br>}}_p\pm <br><br>{\mathrm{\theta <br><br>}}_c\right)}$

and

n_isinθ

_i=n_psin (θ

_p−

θ

_c)

wherein θ

_c is the coupling angle required for light incident at the interface at the base interface of the prism, θ

_i is the incident angle of the light beam on the prism, n_i is the refractive index of the medium at the interface where the light beam enters the prism and n_p is the refractive index of the prism.

6. A method of total internal reflection microscopy according to claim 5, further comprising the step of detecting interaction of a sample at the base of the prism with an evanescent wave formed by total internal reflection of the light beam at the base of the prism.

7. Apparatus for imaging a sample, comprising:

an objective lens having a focal plane and an optical axis, the lens being arranged to collect light from a region of interest defined relative to said optical axis;

an illumination director controllable to direct a beam of light along an optical path so as to illuminate the sample in the region of interest at an angle oblique to said optical axis; and

an illumination director controller arranged to control the illumination director relative to the sample such that the optical path remains directed to the sample in the region of interest following replacement of the sample, or movement of the sample relative to the objective lens.

8. The apparatus of claim 7 further comprising an angular prism having a base and a side face having a base/side face angle θ

_p therebetween, the sample being adjacent to the base of the prism, the beam of light being directed through the side face of the prism at an angle of incidence θ

_i so as to illuminate the sample at the base at a coupling angle θ

_c.

9. The apparatus of claim 8 wherein the base/side face angle θ

_p satisfies the equations;

—

10. The apparatus of claim 9 wherein

θ

_p=θ

_c and θ

_i=0

11. The apparatus of claim 7 wherein the illumination director controller is arranged to adjust the illumination director synchronously with movements parallel to the optical axis of the sample within the region of interest.

12. The apparatus of claim 11 wherein the illumination director controller is arranged such that the position of the illumination director relative to the sample within the region of interest remains substantially constant.

13. The apparatus of claim 7 wherein the illumination director controller is arranged to adjust the illumination director synchronously with movements of the focal plane in a direction parallel to the optical axis and relative to the sample such that the optical path remains directed to the intersection of said region of interest and said focal plane.

14. The apparatus of claim 7 further comprising an autofocus arranged such that the focal plane of the objective lens tracks the sample in the region of interest.

15. The apparatus of claim 14 wherein the autofocus is arranged to:

project an incident range-finding beam of light through the objective lens such that the incident range-finding beam is incident on and reflects from the sample or a surface fixed relative to said sample, to thereby form a reflected range-finding beam collected by said objective lens; and

to detect a displacement of a point of incidence of said reflected range-finding beam in a detection plane.

16. The apparatus of claim 15 arranged such that the incident range-finding beam enters the objective lens displaced from the optical axis.

17. The apparatus of claim 15 wherein the autofocus further comprises a detector and an electrical autofocus coupling to drive the objective lens along the optical axis in response to the displacement of the point of incidence of said reflected range-finding beam in said detection plane.

18. The apparatus of claim 7, wherein at least a part of the objective lens is moveable along the optical axis so as to bring a part of the sample which is within the region of interest into focus, and the illumination director controller comprises a coupling arranged to adjust the illumination director synchronously with the movement of the at least part of the objective along the optical axis.

19. The apparatus of claim 18 wherein the coupling is arranged such that the position of the illumination director relative to that of the at least part of the objective lens remains substantially constant.

20. The apparatus of claim 7 wherein the illumination director controller comprises a direct mechanical coupling.

21. The apparatus of claim 7 wherein the illumination director controller comprises an electrical coupling.

22. The apparatus of claim 7 wherein the objective lens is positioned on a first side of the sample, and the illumination director is positioned on the opposing side of the sample.

23. The apparatus of claim 7 wherein the apparatus is a total internal reflection fluorescence microscope.

24. The apparatus of claim 23 wherein the total internal reflection fluorescence microscope comprises an optic, the sample being situated adjacent to the base of the optic and the illumination beam entering a side face of the optic so as to illuminate the sample, the base/side face angle of the optic being substantially the same as the angle of internal reflection of the illumination beam at the base.

25. The apparatus of claim 7 wherein the illumination director is a first mirror.

26. The apparatus of claim 25 wherein the light beam is directed onto the first mirror by a second mirror, the second mirror being provided with a fine adjust mechanism.

27. A method of automatically correcting the illumination footprint of the optical path of a light beam onto a microscopy sample surface, comprising the step of adjusting the optical path synchronously with changes in the position of the sample surface or the position of an element which moves synchronously with the sample surface.

28. The method of claim 27 wherein the element which moves synchronously with the sample surface is at least a part of an objective lens assembly arranged to form an image of the sample surface.