Light disc microscopy for fluorescence microscopes
View Patent ↗Methods for imaging a sample using fluorescence microscopy, systems for imaging a sample using fluorescence microscopy, and illumination systems for fluorescence microscopes. In some examples, a method includes positioning the sample such that a plane of interest of the sample is coplanar with a focal plane of a detection objective of a microscope. The method includes positioning a paraboloidal mirror around the sample such that a focal point of the paraboloidal mirror is coplanar with the focal plane of the detection objective and the plane of interest of the sample. The method includes directing a beam of annularly collimated excitation light on the paraboloidal mirror to focus a disc of light on the sample and thereby to provide 360 degree lateral illumination of the sample. The method includes imaging the sample through the detection objective.
1. A method for imaging a sample using fluorescence microscopy, the method comprising:
positioning the sample such that a plane of interest of the sample is coplanar with a focal plane of a detection objective of a microscope;
positioning a paraboloidal mirror around the sample such that a focal point of the paraboloidal mirror is coplanar with the focal plane of the detection objective and the plane of interest of the sample;
directing a beam of annularly collimated excitation light on the paraboloidal mirror to focus a disc of light on the sample and thereby to provide 360 degree lateral illumination of the sample; and
imaging the sample through the detection objective.
2. The method of claim 1 , wherein directing the beam of annularly collimated excitation light comprises setting a thickness of the beam of annularly collimated excitation light such that a propagation axis of each coplanar light sheet reflected by the paraboloidal mirror is at an oblique angle relative to the focal plane of the detection objective.
3. The method of claim 2 , wherein setting the thickness of the beam of annularly collimated excitation light comprises increasing the thickness of the beam of beam of annularly collimated excitation light to increase a convergence angle of each coplanar light sheet or decreasing the thickness of the beam of annularly collimated excitation light to decrease the convergence angle of each coplanar light sheet.
4. The method of claim 2 , wherein the detection objective has a numerical aperture greater than or equal to 1.4, wherein the sample is mounted on a glass coverslip, and wherein setting the thickness of the beam of annularly collimated excitation light comprises setting the thickness of the beam of annularly collimated excitation light such that the excitation light does not intersect the glass coverslip before it reaches the sample.
5. The method of claim 1 , wherein directing the beam of annularly collimated excitation light comprises directing a collimated excitation beam onto a conical inner mirror positioned at a center of the collimated excitation beam so that excitation light is symmetrically reflected at every angle about the conical inner mirror.
6. The method of claim 5 , wherein directing the beam of annularly collimated excitation light comprises positioning a conical outer mirror to be centered around the conical inner mirror such that the excitation light that is symmetrically reflected from the conical inner mirror is reflected by the conical outer mirror into the beam of annularly collimated excitation light.
7. The method of claim 6 , wherein the conical outer mirror is a hollow cone of reflective material, and wherein a half angle of the conical outer mirror is complementary to a half angle of the conical inner mirror.
8. The method of claim 1 , wherein positioning the paraboloidal mirror comprises mounting the paraboloidal mirror parallel or antiparallel to the detection objective.
9. The method of claim 1 , wherein imaging the sample comprises observing the sample by eye or camera, using pre-existing light paths within a standard upright or inverted research-grade microscope.
10. The method of claim 1 , wherein imaging the sample comprises performing live-cell fluorescence imaging.
11. A system for imaging a sample using fluorescence microscopy, the system comprising:
a detection objective of a microscope and a sample positioned such that a plane of interest of the sample is coplanar with a focal plane of the detection objective;
a paraboloidal mirror oriented around the sample such that a focal point of the paraboloidal mirror is coplanar with the focal plane of the detection objective and the plane of interest of the sample; and
an illumination system configured for directing a beam of annularly collimated excitation light on the paraboloidal mirror to focus a disc of light on the sample and imaging the sample through the detection objective.
12. The system of claim 11 , wherein directing the beam of annularly collimated excitation light comprises setting a thickness of the beam of annularly collimated excitation light such that a propagation axis of each coplanar light sheet reflected by the paraboloidal mirror is at an oblique angle relative to the focal plane of the detection objective.
13. The system of claim 12 , wherein setting the thickness of the beam of annularly collimated excitation light comprises increasing the thickness of the beam of beam of annularly collimated excitation light to increase a convergence angle of each coplanar light sheet or decreasing the thickness of the beam of annularly collimated excitation light to decrease the convergence angle of each coplanar light sheet.
14. The system of claim 13 , wherein the detection objective has a numerical aperture greater than or equal to 1.4, wherein the sample is mounted on a glass coverslip, and wherein setting the thickness of the beam of annularly collimated excitation light comprises setting the thickness of the beam of annularly collimated excitation light such that the excitation light does not intersect the glass coverslip before it reaches the sample.
15. The system of claim 11 , wherein directing the beam of annularly collimated excitation light comprises directing a collimated excitation beam onto a conical inner mirror positioned at a center of the collimated excitation beam so that excitation light is symmetrically reflected at every angle about the conical inner mirror.
16. The system of claim 15 , wherein directing the beam of annularly collimated excitation light comprises positioning a conical outer mirror to be centered around the conical inner mirror such that the excitation light that is symmetrically reflected from the conical inner mirror is reflected by the conical outer mirror into the beam of annularly collimated excitation light.
17. The system of claim 16 , wherein the conical outer mirror is a hollow cone of reflective material, and wherein a half angle of the conical outer mirror is complementary to a half angle of the conical inner mirror.
18. The system of claim 11 , wherein positioning the paraboloidal mirror comprises mounting the paraboloidal mirror parallel or antiparallel to the detection objective.
19. The system of claim 11 , wherein imaging the sample comprises observing the sample by eye or camera, using pre-existing light paths within a standard upright or inverted research-grade microscope.
20. The system of claim 11 , wherein imaging the sample comprises performing live-cell fluorescence imaging.
21. An illumination system for a fluorescence microscope, the illumination system comprising:
a paraboloidal mirror;
a conical inner mirror;
a conical outer mirror; and
a structure for positioning the conical inner mirror at a center of a collimated excitation beam so that excitation light is symmetrically reflected at every angle about the conical inner mirror, positioning the conical outer mirror to be centered around the conical inner mirror such that the excitation light that is symmetrically reflected from the conical inner mirror is reflected by the conical outer mirror into a beam of annularly collimated excitation light directed at the paraboloidal mirror, and positioning the paraboloidal mirror around a sample such that a focal point of the paraboloidal mirror is coplanar with a focal plane of a detection objective of a microscope and a plane of interest of the sample.