ILLUMINATION ARRANGEMENT FOR TIRF MICROSCOPY
A TIRF illumination having a high axial resolution at low complexity. A TIRF illumination device is designed as a module and comprises an optical fiber and a collimating optic, wherein the collimating optic is mounted in front of a light discharge opening of the optical fiber, such that it collimates light exiting divergently from the optical fiber into a parallel light bundle, such that the excitation light can be applied to a sample outside of the detection beam path. The numerical aperture of the excitation is thus decoupled from the numerical aperture of detection, such that a standard microscope objective is sufficient for detection.
1 . A modular TIRF illumination device for a microscope with an objective lens, the TIRF illumination device comprising:
an optical fiber having a light discharge opening; and
a collimation lens fastened in front of the light discharge opening of the light-wave conduit so that the collimation lens collimates the light emerging divergently from the light-wave conduit to form a light bundle emerging from the collimation lens.
2 . The TIRF illumination device according to claim 1 , wherein the collimation lens is a gradient lens.
3 . The TIRF illumination device according to claim 1 , further comprising a housing for enclosing the collimation lens and at least the light discharge opening of the light-wave conduit.
4 . The TIRF illumination device according to claim 3 , wherein the housing is in sections at the collimation lens.
5 . The TIRF illumination device according to claim 3 , wherein the housing is rod-shaped.
6 . The TIRF illumination device according to claim 5 , wherein the housing is provided with a bearing element.
7 . The TIRF illumination device according to claim 3 , further comprising a glass adapter disposed at the side of the collimation lens pointing away from the light discharge opening.
8 . The TIRF illumination device according to claim 1 , wherein the light-wave conduit comprises one light-conducting fiber.
9 . The TIRF illumination device according to claim 1 , wherein a diameter measured transverse to the optic axis of the collimation lens has a maximum value of 0.7 mm.
10 . The TIRF illumination device according to claim 1 , wherein a focal length of the collimation lens is chosen so that a cross-section of the light bundle corresponds approximately to the diameter of a field of view of the objective lens.
11 . The TIRF illumination device according to claim 1 , wherein the light-wave conduit consists of at least one polarization-preserving, single-mode light-conducting fiber.
12 . A microscope objective for use with a collimation lens and a light-wave conduit, the microscope objective comprising:
an objective having a front lens; and
a support means for the collimation lens and for the light-wave conduit, whereby the collimation lens can be positioned in front of a light discharge opening of the light-wave conduit so that it collimates light emerging divergently from the light-wave conduit to a light bundle, whereby the support means causes the collimated light bundle to cross the optic axes of the objective at an angle that is greater than or equal to the angle of total reflection.
13 . The microscope objective according to claim 12 , further comprising:
a mount for holding the front lens, the support means being formed by a recess in the objective to receive a modular TIRF lighting device consisting of the collimation lens and the light-wave conduit into the mount of the objective and/or in the front lens of the objective.
14 . The microscope objective according to claim 12 , further comprising a connection port for the light-wave conduit, wherein the collimation lens and the connection port are fastened by the support means, whereby the light-wave conduit can be connected removably to the connection port.
15 . The microscope objective according to claim 12 , wherein the support means is installed so that different angles are usable between the light bundle and the optic axis of the microscope objective, and the different angles are each greater than or equal to the total reflection angle.
16 . A method for TIRF excitation in a specimen, the method comprising the steps of:
passing a light through an optical fiber having a light discharge opening and then through a collimation lens fastened in front of the light discharge opening of the light-wave conduit so that the collimation lens collimates the light emerging divergently from the light-wave conduit to form a light bundle emerging from the collimation lens.
17 . The method according to claim 16 , whereby photo-activated localization is performed.
18 . The according to claim 16 , further comprising the steps of passing the collimated light beam as TIRF illumination outside of a detection beam path onto the specimen.
19 . The method according to claim 18 , in which the collimated light beam passes onto the specimen on the same specimen side as the detection beam path.
20 . The method according to claim 18 , in which the collimated light beam passes through a microscope objective.