ILLUMINATION OPTICS FOR AN OPTICAL OBSERVATION DEVICE

Abstract

The invention is directed to illumination optics for an optical device for observing a sample, particularly for TIRF microscopy (Total Internal Reflection Fluorescence Microscopy), wherein the sample is positioned on the side of a carrier glass remote of the illumination optics and the illumination light exiting from the illumination optics is shaped into an illumination beam bundle which encloses an angle not equal to 90° with the normal to the surface of the carrier glass. According to the invention, the illumination optics of the type mentioned above comprise at least two optically active elements which influence the shape and direction of the illumination beam bundle and which are arranged outside of the detection beam path that guides light coming from the sample to a detector. The optically active elements are preferably constructed as annular lenses and are arranged concentrically around the detection beam path.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 illustrates illumination optics in accordance with the invention in diagrammatic form; and

FIG. 2 illustrates an enlarged view of area A of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows illumination optics, according to the invention, which comprise four annular lenses 1 to 4. Lens 4 forms the front lens from which the illumination beam bundle 5 exits and strikes the interface between a carrier glass 6 and the sample 7 at an angle β which satisfies the condition of total reflection. An immersion liquid 8 is provided between the front lens 4 and the carrier glass 6.

The light coming from the sample 7 travels in a detection beam path 9 in the cut out center of the annular lenses 1 to 4 and reaches a detection device, not shown.

Lenses 1 to 4 have the radii r, thicknesses d, distances a in mm, refractive indexes n_e at wavelength 546 nm, Abbe numbers v_e, and free diameters Frd indicated in the following table. Contrary to the positive distances between the lenses which are otherwise only possible and conventional, negative distances or the distance 0 mm are also possible. This is easy to understand: if the drilled lens were filled to form a normal lens, the lenses would penetrate one another because of the very presence of this negative distance of the lens vertexes.

					Refractive	Abbe
Lens	Surface	Radius r	Thickness d	Distance a	index ne	number νe	Frd
1	2	−25.0000	5.28456		1.52458	59.22	28
	3	−28.1068
				12.4776
2	4	22.7704	8.12534		1.48914	70.23	30
	5	−3033.99
				0.0000
3	6	10.4645	8.0000		1.48914	70.23	20.6
	7	12.9410					15.0
				−5.0000
4	8	6.6167		10.5253	1.52458	59.22	13.2334
	9	plane

NK5 and NFK5 are possible types of glass that may be used. The carrier glass 6 is planar and has a thickness of 0.17 mm.

Standard immersion oil with a refractive index n_e=1.518 and a dispersion v_e=47.37 is advantageously used as immersion liquid 8.

The angle β is varied by changing the distance from the focus point to the optical axis. For example, when the distance between the focus point and the optical axis is 12.5 mm, angle β is 82.5°; when this distance is 12.17 mm, angle β is 74.1°, etc.

FIG. 2 shows a larger view of the area A indicated in FIG. 1. It can be seen that the illumination beam path 5 exits from the front lens 4 as a parallel light bundle and is directed through the immersion liquid 8 and the carrier glass 6 to the interface between the side of the carrier glass 6 remote of the front lens 4 and the sample 7.

Fields with a diameter of about 580 μm can be observed on the sample with this construction of the illumination optics according to the invention. This results in a significant advantage over the prior art because previously only fields of about 110 μm diameter could be observed.

This advantage is achieved substantially because separate illumination optics are used which can have a different focal length than the detection optics.

Another advantage is the possibility of reducing the penetration depth because angles β up to 81.2° can be realized with the illumination optics according to the invention, which corresponds to a numerical aperture of 1.50 when the refractive index of the immersion oil is 1.518. Because of the available long focal length by which the light source is imaged in the sample, it possible to vary the illumination angle or angle β substantially more accurately than in the prior art in which the illumination of the sample is carried out through a microscope objective.

Another substantial advantage consists in that the autofluorescence of the material from which the optical elements of the microscope objective are made in the prior art need not be taken into account because these elements are no longer penetrated by the illumination light (which corresponds to the excitation light in fluorescence microscopy).

Therefore, the entire optical system of these illumination optics can be optimized specifically to the shorter wavelengths of fluorescence excitation radiation, and it is now only necessary to correct the outside pupil area for a light bundle with a small diameter so that the optical system can be designed in an uncomplicated manner.

While the foregoing description and drawings represent the present invention, it will be obvious to those skilled in the art that various changes may be made therein without departing from the true spirit and scope of the present invention.

REFERENCE NUMBERS

• 1, 2, 3 lenses
• 4 front lens
• 5 illumination beam bundle
• 6 carrier glass
• 7 sample
• 8 immersion liquid
• 9 detection beam path
• β angle

Claims

1. Illumination optics for an optical device for observing a sample, particularly for TIRF microscopy (Total Internal Reflection Fluorescence Microscopy), wherein a sample is arranged on a side of a carrier glass remote of the illumination optics and illumination light exiting from the illumination optics is shaped into an illumination beam bundle which encloses an angle not equal to 90° with the normal to the surface of the carrier glass, comprising: at least two optically active elements which influence the shape and direction of the illumination beam bundle and which are arranged outside of the detection beam path that guides light coming from the sample to a detector.

2. Illumination optics according to claim 1, wherein the optical elements are annular and are arranged concentrically around the detection beam path.

3. Illumination optics according to claim 1, wherein the optically active elements are constructed as annular lenses with light entrance surfaces and light exit surfaces arranged concentrically around the detection beam path.

4. Illumination optics according to claim 3, wherein an immersion liquid is provided between the light exit surface of the final lens in direction of the illumination beam path, i.e., the front lens, and the carrier glass.

5. Illumination optics according to claim 4, wherein the glass from which the front lens is made has a refractive index ne that diverges from the refractive index ne of immersion liquid by no more than 0.05 and whose dispersion ve diverges from the dispersion ve of the immersion liquid by no more than 20.

6. Illumination optics according to claim 5, comprising four lenses having the radii r, thicknesses d, distances a in mm, refractive indexes ne at wavelength 546 nm, Abbe numbers ve, and free diameters Frd indicated in the following table:

7. Illumination optics according to claim 6, wherein the illumination beam path exits from the front lens as a parallel light bundle, wherein all of the beam components of the parallel light bundle which differ with respect to wavelength strike the interface between the carrier glass and sample at the same reflection angle β.

8. Illumination optics according to claim 1, wherein the optical elements are constructed as annular mirrors with mirror surfaces arranged concentrically around the detection beam path.

9. Illumination optics according to claim 8, wherein at least one of the mirrors is constructed as a rear-surface mirror.

10. Illumination optics according to claim 8, wherein the final mirror before the carrier glass considered in direction of the illumination beam path, i.e., the front mirror, is constructed as a rear-surface mirror, and an immersion liquid is provided between the front mirror and the carrier glass.

11. Illumination optics according to claim 1, wherein optical elements which are constructed as annular lenses and as annular mirrors are provided.

12. Illumination optics according to claim 1, with a focal length in the range of 1 mm to 50 mm.

13. Illumination optics according to claim 1, wherein the angle β can be up to 81.2°, which corresponds to a numerical aperture of 1.50 when the refractive index ne of the immersion media is 1.518.

14. A method of using illumination optics according to claim 1 in a microscope with an incident light illumination system.

15. A method of using illumination optics according to claim 1 in a microscope with a transmitted light illumination system