Method for calibrating a deflection unit in a TIRF microscope, TIRF microscope, and method for operating the same
View Patent ↗The invention relates to a method for calibrating a deflection unit in a TIRF microscope, by means of which an angle of incidence of excitation light onto a specimen is adjusted, wherein a setting of said deflection unit is adjusted such that the pertaining angle of incidence is definitely greater or definitely smaller than an anticipated critical angle for total reflection of the excitation light on a surface of a used specimen, the angle of incidence is scanned by varying the setting of said deflection unit in the direction of an anticipated critical angle, an intensity of an optical response of the used specimen elicited by the excitation light being measured for each setting of the deflection unit, the intensity of the optical response of the specimen used is measured at least for a number of settings of the deflection unit until the intensity of the optical response of the specimen used traverses a flank, and the setting of the deflection unit pertaining to the flank is stored as the setting for the critical angle for total reflection at the specimen used.
1. A method for calibrating a deflection unit in a TIRF microscope, with which an angle of incidence of excitation light onto a specimen is adjusted, comprising:
(a) adjusting a setting of the deflection unit such that a pertaining angle of incidence is greater or smaller than an anticipated critical angle for total reflection of an excitation light on a surface of a specimen;
(b) scanning the angle of incidence by varying the setting of the deflection unit in a direction of the anticipated critical angle, and measuring an intensity of an optical response of a specimen elicited by the excitation light for each setting of the deflection unit;
(c) measuring the intensity of the optical response of the specimen at least for a plurality of different settings of the deflection unit until the intensity of the optical response of the specimen traverses a flank; and
(d) storing the setting of the deflection unit pertaining to the flank as the setting for the critical angle for total reflection at the specimen.
2. The method as defined in claim 1 , wherein the optical response of the specimen is measured in a plane conjugated to a plane of an objective pupil.
3. The method as defined in claim 1 , wherein the optical response is measured with a detector.
4. The method as defined in claim 1 , wherein the optical response is measured with a camera.
5. The method as defined in claim 4 , wherein the optical response is measured with a camera used in a TIRF measuring mode.
6. The method as defined in claim 4 , wherein, for evaluation of the intensity, only subregions of a camera detector are used.
7. The method as defined in claim 4 , wherein, for acceleration of the calibration of the angle of incidence, the camera is operated at a reduced resolution.
8. The method as defined in claim 1 , wherein, for acceleration of a calibration of the angle of incidence, scanning is effected only in a vicinity of the anticipated critical angles or of an anticipated critical angle.
9. The method as defined in claim 1 , wherein, to achieve a punctiform pupil illumination, a first setting of the deflection unit is determined for a positive critical angle for total reflection and a second setting of the deflection unit is determined for a negative critical angle for total reflection, and wherein the setting of the deflection unit for a vertical incidence of the excitation light onto the surface of the specimen is defined as a midpoint between the first setting and the second setting.
10. The method as defined in claim 1 , wherein the optical response of the specimen is measured in a plane conjugated to a plane of the surface of the specimen.
11. The method as defined in claim 1 , wherein, as optical response, at least one of a fluorescent light or a scattered light of the specimen is measured.
12. The method as defined in claim 1 , wherein, to achieve a punctiform pupil illumination using refractive indices of the specimen and of a cover glass, and following determination of the settings of the critical angle for total reflection, unique absolute values for the angle of incidence are assigned to the settings of the deflection unit.
13. The method as defined in claim 1 , wherein, to achieve a circularly annular pupil illumination utilizing refractive indices of the specimen and of the cover glass, and following determination of the setting of the critical angle for total reflection, unique absolute values for the angle of incidence are assigned to the settings of the deflection unit.
14. A method for operating a TIRF microscope, the TIRF microscope comprising a light source for the emission of excitation light; microscope optics for guiding the excitation light onto the specimen; a deflection unit for the adjustment of the angle of incidence of the excitation light on the surface of the specimen; a detector for detecting the optical response of the specimen; and supplementary optics, capable of being revolved into an optical imaging path, for switching between an imaging of the specimen and an imaging of an objective pupil on the detector;
wherein the deflection unit is calibrated by the method defined in claim 1 , the angle of incidence of the excitation light is adjusted to a value, the magnitude of which is greater than a critical angle for total reflection, and TIRF measurements are then carried out.
15. The method as defined in claim 14 , wherein on the basis of the mathematical relationship:
d
=
λ
4
π
·
n
d
sin
2
α
-
sin
2
α
T
in which
λ denotes a wavelength used,
n d denotes a refractive index of a cover glass,
α is the angle of incidence of the excitation light on the specimen, and
α T is the critical angle for total reflection,
the associated angle of incidence is adjusted to reach a desired depth of penetration d of an evanescent field in the specimen.
16. The method as defined in claim 15 , wherein the refractive index of the specimen is determined by utilizing the critical angle for total reflection and the refractive index of the cover glass.
17. A TIRF microscope, comprising:
a light source for the emission of excitation light;
microscope optics for guiding the excitation light onto the specimen;
a deflection unit for the adjustment of the angle of incidence of the excitation light on the surface of the specimen, wherein the deflection unit is configured for:
adjusting a setting of the deflection unit such that a pertaining angle of incidence is greater or smaller than an anticipated critical angle for total reflection of an excitation light on a surface of a specimen;
scanning the angle of incidence by varying the setting of the deflection unit in a direction of the anticipated critical angle, and measuring an intensity of an optical response of a specimen elicited by the excitation light for each setting of the deflection unit;
measuring the intensity of the optical response of the specimen at least for a plurality of different settings of the deflection unit until the intensity of the optical response of the specimen traverses a flank; and
storing the setting of the deflection unit pertaining to the flank as the setting for the critical angle for total reflection at the specimen;
a detector for detecting the optical response of the specimen; and
supplementary optics, capable of being revolved into an optical imaging path, for switching between an imaging of the specimen and an imaging of an objective pupil on the detector.
18. The TIRF microscope as defined in claim 17 , wherein a separate detector is provided for measuring the optical response.
19. A TIRF microscope, comprising:
a light source for the emission of excitation light;
microscope optics for guiding the excitation light onto the specimen;
a deflection unit for the adjustment of the angle of incidence of the excitation light on the surface of the specimen, wherein the deflection unit is configured for:
adjusting a setting of the deflection unit such that a pertaining angle of incidence is greater or smaller than an anticipated critical angle for total reflection of an excitation light on a surface of a specimen;
scanning the angle of incidence by varying the setting of the deflection unit in a direction of the anticipated critical angle, and measuring an intensity of an optical response of a specimen elicited by the excitation light for each setting of the deflection unit;
measuring the intensity of the optical response of the specimen at least for a plurality of different settings of the deflection unit until the intensity of the optical response of the specimen traverses a flank; and
storing the setting of the deflection unit pertaining to the flank as the setting for the critical angle for total reflection at the specimen;
a detector for detecting the optical response of the specimen; and
a separate detector for measuring the optical response.
20. The TIRF microscope as defined in claim 19 , further comprising:
supplementary optics, capable of being revolved into an optical imaging path, for switching between an imaging of the specimen and an imaging of an objective pupil on the detector.