Total internal reflection fluorescence microscope (TIRFM)

Patent metadata

Field	Value
Publication number	US-9964749-B2
Application number	US-201615339091-A
Country	US
Kind code	B2
Filing date	Oct 31, 2016
Priority date	Nov 30, 2015
Publication date	May 8, 2018
Grant date	May 8, 2018

Abstract

Disclosed is a fluorescence microscope for imaging a specimen containing a fluorescent substance, the fluorescence microscope including an excitation light source configured to emit an excitation light that excites a fluorescent substance to emit fluorescence; a de-excitation light source configured to emit a de-excitation light that de-excites the fluorescent substance excited by the excitation light emitted from the excitation light source; an optical body configured to overlap a light emitted from the excitation light source and a light emitted from the de-excitation light source, and to discharge the overlapped light toward the specimen; and a solid immersion lens to which the light discharged from the optical body is incident, and configured to refract the light discharged from the optical body toward the specimen. A total internal reflection of the light incident to the solid immersion lens occurs on a bottom of the solid immersion lens.

First claim

What is claimed is:

1. A total internal reflection fluorescence microscope (TIRFM) for imaging a specimen containing a fluorescent substance, the TIRFM comprising:
an excitation light source configured to emit an excitation light that excites the fluorescent substance to emit fluorescence;
a de-excitation light source configured to emit a de-excitation light that de-excites the fluorescent substance excited by the excitation light emitted from the excitation light source;
an optical body configured to overlap a light emitted from the excitation light source and a light emitted from the de-excitation light source, and to discharge the overlapped light toward the specimen; and
a solid immersion lens to which the light discharged from the optical body is incident, and configured to refract the light discharged from the optical body toward the specimen,
wherein a total internal reflection of the light incident to the solid immersion lens occurs on a bottom of the solid immersion lens.

2. The TIRFM of claim 1 , further comprising:
an aperture configured to cover a portion of the excitation light emitted from the excitation light source.

3. The TIRFM of claim 2 , wherein the aperture is configured to block a light of an area on which a total internal reflection does not occur on the bottom of the solid immersion lens in the excitation light incident to the solid immersion light by blocking a portion of the excitation light emitted from the excitation light source.

4. The TIRFM of claim 3 , wherein the aperture is configured to block a light of an area corresponding to a threshold angle or less in the excitation light incident to the solid immersion light by blocking a portion of the excitation light emitted from the excitation light source.

5. The TIRFM of claim 2 , wherein the aperture comprises:
a penetrator formed of a transparent material, and configured to allow the excitation light emitted from the excitation light source to pass; and
a shield formed of an opaque material, and configured to block the excitation light discharged from the excitation light source, and
the penetrator and the shield are provided so that an incidence angle of the excitation light incident to the solid immersion lens is greater than a threshold angle.

6. The TIRFM of claim 5 , wherein the shield is provided in a circular shape to block a center of the excitation light emitted from the excitation light source, and the penetrator is provided in a ring shape around the shield.

7. The TIRFM of claim 1 , further comprising:
a detector configured to detect a fluorescent light from the fluorescent substance of the specimen.

8. The TIRFM of claim 1 , wherein the optical body comprises:
an excitation light transmitter configured to transfer the excitation light emitted from the excitation light source toward the specimen; and
a de-excitation light transmitter configured to transfer the de-excitation light emitted from the de-excitation light source toward the specimen.

9. The TIRFM of claim 8 , wherein the excitation light transmitter and the de-excitation light transmitter are dichroic mirrors or beam splitters configured to reflect the excitation light or the de-excitation light, and to allow a fluorescent light emitted from the fluorescent substance of the specimen to pass.

10. The TIRFM of claim 1 , wherein an object lens configured to allow the light discharged from the optical body to be incident toward the solid immersion lens is provided below the optical body.

11. The TIRFM of claim 1 , wherein, in response to the excitation light being incident to the solid immersion lens at an angle greater than a threshold angle, a total internal reflection occurs on the bottom of the solid immersion lens, an evanescent wave toward the specimen occurs on the solid immersion lens, and a fluorescent light of the fluorescent substance generated by the evanescent wave is detected.

12. The TIRFM of claim 1 , wherein pieces of the de-excitation light are formed in a donut shape to overlap the excitation light on a neighboring area excluding a central area of the excitation light, and
a horizontal resolution increases in response to an occurrence of a simulated emission depletion (STED) phenomenon that de-excites the excitation light for exciting the fluorescent substance.

13. The TIRFM of claim 10 , wherein the solid immersion lens is provided in a hemispherical shape, and the light discharged from the object lens is incident to be vertical to the surface of the solid immersion lens and to increase a light collecting efficiency.

14. The TIRFM of claim 13 , further comprising:
a replicated lens configured to refract the light discharged from the optical body on the surface of the solid immersion lens or the object lens.

15. The TIRFM of claim 13 , wherein the bottom of the solid immersion lens is provided in a conic shape that is upwardly inclined with getting closer from a center to an edge.

16. A total internal reflection fluorescence microscope (TIRFM) for imaging a specimen containing a fluorescent substance, the TIRFM comprising:
an excitation light source configured to emit a first wavelength of an excitation light that excites the fluorescent substance;
a de-excitation light source configured to emit a second wavelength of a de-excitation light that de-excites the fluorescent substance excited by the excitation light emitted from the excitation light source;
an optical body configured to overlap a light emitted from the excitation light source and a light emitted from the de-excitation light source, and to discharge the overlapped light toward the specimen;
a solid immersion lens to which the light discharged from the optical body is incident, and configured to refract the light discharged from the optical body toward the specimen;
an aperture configured to adjust an amount of light emitted from the excitation light source by covering at least a portion of the excitation light source and to enable the light discharged from the optical body to cause a total internal reflection on a bottom of the solid immersion lens; and
a detector configured to collect a fluorescent reaction of the fluorescent substance that has received the light discharged from the optical body.

17. The TIRFM of claim 16 , wherein the aperture is configured to block a light of an area on which the total internal reflection does not occur on the bottom of the solid immersion lens in the excitation light incident to the solid immersion lens by blocking a portion of the excitation light emitted from the excitation light source.

18. The TIRFM of claim 17 , wherein the aperture is configured to block a light of an area corresponding to a threshold angle or less in the excitation light incident to the solid immersion light by blocking a portion of the excitation light emitted from the excitation light source.

19. The TIRFM of claim 16 , wherein the aperture comprises:
a penetrator formed of a transparent material, and configured to allow the excitation light emitted from the excitation light source to pass; and
a shield formed of an opaque material, and configured to block the excitation light discharged from the excitation light source, and
the penetrator and the shield are provided so that an incidence angle of the excitation light incident to the solid immersion lens is greater than a threshold angle.

20. The TIRFM of claim 16 , wherein the shield is provided in a circular shape to block a center of the excitation light emitted from the excitation light source,

Assignees

• Univ Yonsei Iacf

Inventors

• Park No Cheol
• Moon Hyung Bae
• Lee Won Sup
• Lim Geon
• Choi Guk Jong

Classifications

• G02B21/33

  Immersion oils {, or microscope systems or objectives for use with immersion fluids} · CPC title

• G02B21/082

  for incident illumination only · CPC title

• G02B21/086

  for transillumination only · CPC title

• G02B21/361

  Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups) · CPC title

• G02B21/16 Primary

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title