EP1668395A1

EP1668395A1 - Lens for evanescent wave illumination and corresponding microscope

Info

Publication number: EP1668395A1
Application number: EP04787201A
Authority: EP
Inventors: Heinrich Ulrich; Werner Knebel; Kyra MÖLLMANN
Original assignee: Leica Microsystems Heidelberg GmbH; Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2003-09-25
Filing date: 2004-09-23
Publication date: 2006-06-14
Also published as: WO2005029150A1; US20080266659A1; US8817368B2

Abstract

The invention relates to a lens (1) for total internal reflection microscopy. The lens (1) is characterised in that a light source (5, 7) and/or at least one deviating means (31, 3) which deviates the light from a light source (5, 7) into the lens and/or the emitting end (35) of an illuminating optical fibre (37) is arranged in the region of the rear focal plane (3) of the lens (1).

Description

Lens for evanescent lighting and microscope

The invention relates to a lens for total internal reflection microscopy.

The invention also relates to a microscope for total internal reflection microscopy with a light source and with an objective

A microscope with evanescent illumination of a sample is known from US 2002/0097489 A1. The microscope contains a white light source, the light of which is coupled via a slit diaphragm through the microscope objective into the specimen slide for evanescent illumination. The illuminating light propagates in the slide by total internal reflection, the sample being illuminated only in the area of the evanescent field protruding from the slide. Microscopes of this type are known under the term TIRFM (Total Internal Reflection Fluorescent Microscope). The z-resolution of TIRF microscopes is extremely good due to the evanescent field that only projects into the sample by approx. 100 nm.

DE 101 08 796 A1 discloses a high-aperture lens, in particular for TIRF applications. The objective consists of a first lens with a positive refractive power, a second lens with a negative refractive power, whereby the focal length ratio between the two lenses is in the range of - 0.4 and - 0.1 and the total refractive power is greater than zero. Furthermore, the lens contains two positive lenses, whose ratio diameter to the focal length is greater than 0.3 and less than 0.6. The objective also includes a negative lens and a converging lens, the negative lens facing the front group and the focal length ratio of the negative lens and the converging lens being between −0.5 and −2.

From DE 102 17 098 A1 an incident light arrangement for TIRF microscopy is known. The incident light illuminating arrangement contains an illuminating source that is polarized during operation

Outputs illuminating beam that propagates at an angle to the optical axis and a deflection device that deflects the illuminating beam and couples it into the lens parallel to the optical axis. It is provided in this incident light lighting arrangement that the illumination beam emitted by the illumination source has s and p polarization directions with a phase difference and the deflection device reflects the illumination beam x times, where x = (n x 180 ° - d) / 60 °.

A microscope for TIRM (Total Internal Reflection Microscopy) is known from DE 101 43 481 A1. The microscope has a microscope housing and an objective. The illuminating light emanating from an illuminating device can be coupled in via an adapter which can be inserted into the microscope housing.

From US 2004/0001253 A1 is a microscope with an optical lighting system that enables simple switching between evanescent lighting and reflection lighting. The

Illumination system includes a laser light source, the light of which is coupled into an optical fiber. A coupling optic is also provided, which focuses the light emerging from the fiber into a rear focal point of the microscope objective. The optical fiber can be displaced in a plane perpendicular to the optical axis of the microscope objective. DE 102 29 935 A1 discloses a device for coupling light into a microscope. Laser light is directed onto the specimen in the light field diaphragm plane by an optical fiber coupling designed as a slide. The invention is particularly suitable for the TIRF method.

In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted by the sample, as reflection or fluorescent light. The focus of one

Illumination light beams are moved in a sample plane with the aid of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another, so that one mirror deflects in the x and the other in the y direction. The mirrors are tilted, for example, with the help of galvanometer control elements. The power of the detection light coming from the object is measured depending on the position of the scanning beam. The control elements are usually equipped with sensors for determining the current mirror position. Especially in confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, focusing optics with which the light from the source is focused on a pinhole - the so-called excitation diaphragm - a beam splitter, a beam deflection device for beam control, microscope optics, a detection diaphragm and the detectors for detecting the detection - or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflection light coming from the object reaches the beam splitter via the beam deflection device, passes it, and is then focused on the detection diaphragm behind which the detectors are located. This detection arrangement is called a descan arrangement. Detection light that does not originate directly from the focus region takes a different light path and does not pass through the detection diaphragm, so that one obtains point information that passes through sequential scanning of the object with the focus of the illuminating light beam leads to a three-dimensional image. A three-dimensional image is usually achieved by taking image data in layers. The systems known from the prior art have the disadvantage that in some cases very complex and space-consuming optics are necessary in the beam path of the microscope in order to couple the TIRF illuminating light. This adversely affects the detection beam path in particular and often leads to a loss of detection light output. It is the object of the present invention to provide a lens for use in total internal reflection microscopy, which enables the TIRF illuminating light to be coupled into the beam path of the microscope in a particularly efficient manner.

This object is achieved by a lens, which is characterized in that a light source and / or at least one deflection means, which directs the light from a light source into the lens, and / or the exit end of an illuminating optical fiber is arranged in the region of the rear focal plane of the lens.

It is a further object of the present invention to provide a microscope in which the coupling of the TIRF illuminating light is possible efficiently and with little loss and which at the same time largely makes additional optics in the beam path of the microscope superfluous.

The further object is achieved by a microscope, which is characterized in that, in the region of the rear focal plane of the objective, the light source and / or at least one deflection means, which directs the light from the light source into the objective, and / or the exit end of an illuminating optical fiber, which is fed by the light source, is arranged.

The invention has the advantage that no beam splitters in the beam path of the microscope are necessary for coupling in the TIRF illuminating light. This enables an increased fluorescence yield - since no signal loss is caused. In addition, a high Time resolution can be achieved since no optical components, in particular no beam splitters, have to be switched.

In a preferred embodiment, the light source and / or at least the deflection means and / or the exit end of the illuminating light fiber is arranged directly in the rear focal plane of the objective.

Almost any light source customary in microscopy can be used as the light source. The light source preferably comprises at least one laser, which can be configured, for example, as a semiconductor laser and / or contains at least one LED (light-imitting diode). In a special embodiment variant, the light source can comprise several individual light sources; the individual light sources are preferably arranged in a ring. The ring of individual light sources can be arranged, for example, in the lens housing itself, but also on the outside of the lens housing, openings being provided for coupling light. If the individual light sources are arranged outside the lens housing, a deflection means is preferably arranged in the rear focal plane or in the region of the rear focal plane of the objective, which deflects the light of the individual light sources in such a way that it emerges from the front lens of the lens at the desired angle. In a particularly preferred embodiment variant, the light source comprises a plurality of individual light sources which emit illuminating light of different wavelengths. The individual light sources can preferably be switched on and off independently of one another.

In a preferred embodiment of the microscope according to the invention, a detector, in particular a multiband detector or a spectrometer, is provided which detects synchronously with the switching on of the light source and / or the individual light sources. This variant is particularly advantageous in the case of samples which contain, for example, a plurality of fluorescent dyes, because, according to the invention, spectral analysis of the sample is possible very quickly.

According to the invention, it is possible to use any excitation light wavelengths to select or select different combinations of excitation wavelengths and switch the individual light sources simultaneously or sequentially accordingly.

In a variant of the objective according to the invention or of the microscope according to the invention, optics are provided which focus the light from the light source onto the deflection means.

In a preferred embodiment variant, an at least partial coating of an (already present) objective element, for example a lens, forms the deflection means. This can be a partially reflective or completely reflective coating. In a particularly preferred variant, the coating is designed as a dielectric mirror coating, the coating being designed such that the detection light can penetrate it largely unhindered.

After passing through the objective, the light of the light source preferably emerges at least at an adjustable angle to the optical axis

Lens off. This angle is crucial for the evanescent depth of penetration into the sample. The position of the light source and / or the at least one deflecting means and / or the exit end is preferably the

Optical fiber can be changed within the focal plane. The position of the light source or the deflecting means or the exit end directly influences the angle to the optical axis at which the light from the light source follows

Passing through the lens emerges from the lens. To set the

Position of the light source or the deflecting means or the exit end can be used mechanical adjusting elements, as are common in microscopy. In a special variant, the angle is for

Illumination light of different wavelengths each different.

In a special embodiment variant, the microscope comprises a scanning microscope, in particular a confocal scanning microscope. A microscope designed as a confocal scanning microscope is particularly suitable for carrying out experiments on the basis of photoactivation or caged-combound release, and in doing so at the TIRF level observe. With such an arrangement, the time resolution is particularly large since, for example, sample stimulation and camera observation can be carried out simultaneously.

The microscope according to the invention is particularly suitable for carrying out biological experiments, in particular for photoactivation and / or caged combound release and / or FRAP (fluorecence recovery after photobleaching).

Bandpass filters and / or edge filters are preferably arranged in front of the detector, which can be configured as a camera, for example, to be matched to the respective emission focal length of the fluorescence signal. A dispersive element can be provided for color selection, which generates a spectral splitting from which the wavelength components to be detected are masked out. The detector can also be designed as a color detector, for example as a color camera. It is also possible for a dispersive element to split the detection light over several detectors in order to achieve spectral detection.

The subject matter of the invention is shown schematically in the drawing and is described below with reference to the figures, elements having the same effect being provided with the same reference symbols. 1 shows an objective according to the invention

Fig. 2 Another lens according to the invention

Fig. 3 Another lens according to the invention

Fig. 4 An inventive microscope and

Fig. 5 Another microscope according to the invention

1 shows an objective 1 according to the invention, which can be used for total internal reflection microscopy. A light source 5 is arranged in the rear focal plane 3 of the objective 1 and is formed from a first individual light source 7 and a second individual light source 9. The first individual light source 7 comprises a light-emitting diode (LED), while the second individual light source includes a semiconductor laser. The individual light sources 7, 9 emit illuminating light 11, 13 of different wavelengths. The objective comprises a front lens 15 and a further lens 17. The illuminating light 11 or illuminating light 13 emerges from the microscope objective at an angle Betta or at an angle alpha to the optical axis 19. The angle can be adjusted by moving the individual light source 7 or by moving the individual light source 9 within the rear focal plane. For this purpose, the distance of the individual light source 7 or of the further individual light source 9 to the optical axis is changed using a mechanical adjusting device, not shown. The lens 1 has a lens housing 21.

FIG. 2 shows a further lens 1 according to the invention with a lens housing 21 which has a first opening 23 and a further opening 25. The objective has individual light sources 27, 29 arranged in a ring, which are arranged in a ring and belong to a light source 5. The light source 5 is arranged outside the lens housing 21 and the light from the light source 5 is coupled in via the first opening 23 and the second opening 25 and via further openings (not shown) in the lens housing 21. For coupling, deflection means 31, 33 are provided in the area of the rear focal plane 3 of the objective 1, which deflect the light from the light source 5 into the further beam path of the objective 1.

3 shows a further objective according to the invention with a lens housing 21, behind the rear focal plane 3 of which the exit end 35 of an illuminating optical fiber 37 is arranged. The illuminating light 39 of a light source 5, which is designed as a laser 41, is coupled into the illuminating optical fiber 37 with the aid of a coupling optics 43. The distance between the exit end 35 of the illumination optical fiber 37 and the optical axis 19 of the objective 1 can be adjusted mechanically. For this purpose, the exit end of the illuminating optical fiber is arranged to be movable within the rear focal plane 3 of the objective 1. The illuminating light 45 emerging from the illuminating optical fiber 37 leaves the objective 21 at an angle alpha to the optical axis 19. This angle can be adjusted by adjusting the position of the exit end 35 of the illumination optical fiber 37 within the rear focal plane 3 of the objective 1.

FIG. 4 shows a microscope according to the invention with an objective 1, as was described in FIG. 2. The illuminating light 47 emanating from the individual light source 27 illuminates the sample 51 arranged on the cover glass 49 in an evanescent manner. The detection light 53 emanating from the sample passes through the microscope objective 1 to the tube lens 55 and is then directed by the mirror 57 and via the optics 59 to the detector 61, which is designed as a CCD camera 63. A filter wheel 65 with several different individual filters for selecting the detection spectral range is arranged in front of the detector 61.

FIG. 5 shows a further microscope according to the invention, which is similar to the microscope described with reference to FIG. 4. In addition, a confocal scanner 67 is provided, the scanning light beam 69 of which is coupled through the scanning lens 71 via the beam splitter 73 into the beam path of the microscope. The scanning light beam 69, which is shown in dashed lines in the figure, reaches the sample 51 through the tube lens and the microscope objective. The scanning light beam 69 can, for example, be used to excite the sample in addition to the evanescent field and / or to manipulate the sample. Simultaneously to record an image of the sample with the detector 61, a confocal raster image recording of the sample can also take place, with the confocal scanner 67 detection light 75 (drawn in dashed lines) emanating from the sample, which reaches the confocal scanner 67 in the opposite light path as the scan beam 69 , receives.

The invention has been described in relation to a particular embodiment. However, it is understood that changes and modifications can be made without departing from the scope of the following claims. LIST OF REFERENCE NUMBERS

1 lens

3 rear focal plane

5 light source

7 first individual light source

9 second single light source

11 illuminating light

13 illuminating light

15 front lens

17 more lenses

19 optical axis

21 lens housing

23 first opening

25 more openings

27 single light source

29 individual light source 1 deflecting means

33 deflecting means 5 exit end 7 illuminating optical fiber 9 illuminating light 1 laser 3 coupling optics 5 illuminating light 47 Illumination light

49 coverslip

51 rehearsal

53 detection light

55 tube lens

57 mirrors

59 optics

61 detector

63 camera

65 filter wheel

67 scanners

69 scanning light beam

71 Scan lens

73 beam splitter

75 detection light

Claims

claims

1. Objective for Totaliπterneπ reflection microscopy, characterized in that in the region of the rear focal plane of the objective a light source and / or at least one deflection means, which directs the light from a light source into the objective, and / or the exit end of an illuminating optical fiber is arranged.

2. Lens according to claim 1, characterized in that the light source comprises at least one laser, in particular a semiconductor laser, and / or at least one LED.

3. Lens according to one of claims 1 or 2, that the light source comprises a plurality of individual light sources which are arranged in a ring.

4. Lens according to one of claims 1 to 3, characterized in that the light source comprises a plurality of individual light sources which emit illuminating light of different wavelengths.

5. Lens according to one of claims 3 or 4, characterized in that the individual light sources can be switched on and off independently of one another.

6. Lens according to one of claims 1 to 5, characterized in that an optical system is provided which focuses the light of the light source on the deflecting means.

7. Lens according to one of claims 1 to 6, characterized in that a partial coating of a lens element, in particular a lens, forms the deflecting means.

8. Lens according to claim 7, characterized in that the partial coating comprises a, preferably dielectric, mirror coating.

9. Lens according to one of claims 1 to 8, characterized in that the light of the light source after passing through Lens emerges from the lens at at least one adjustable angle to the optical axis.

10. Lens according to claim 9, characterized in that the position of the light source and / or the at least one deflecting means and / or the outlet end can be changed within the focal plane.

11. Lens according to one of claims 9 or 10, characterized in that the angle for illuminating light of different wavelength is different.

12. microscope for total internal reflection microscopy with a light source and with a lens, characterized in that in the region of the rear focal plane of the lens, the light source and / or at least one deflecting means that directs the light from the light source into the lens, and / or the exit end of an illuminating optical fiber, which is fed by the light source, is arranged.

13. Microscope according to claim 12, characterized in that the

Light source comprises at least one laser, in particular a semiconductor laser, and / or at least one LED.

14. Microscope according to one of claims 12 or 13, that the light source comprises a plurality of individual light sources which are arranged in a ring.

15. Microscope according to one of claims 12 to 14, characterized in that the light source comprises a plurality of individual light sources which emit illuminating light of different wavelengths.

16. Microscope according to one of claims 14 or 15, characterized in that the individual light sources can be switched on and off independently of one another.

17. Microscope according to one of claims 12 to 16, characterized in that an optical system is provided which focuses the light from the light source onto the deflection means.

18. Microscope according to one of claims 12 to 17, characterized characterized in that a partial coating of an objective element, in particular a lens, forms the deflecting means.

19. Microscope according to claim 18, characterized in that the partial coating comprises a, preferably dielectric, mirror coating.

20. Microscope according to one of claims 12 to 19, characterized in that the light of the light source emerges from the lens after passing through the lens at at least one adjustable angle to the optical axis.

21. Microscope according to claim 20, characterized in that the

Position of the light source and / or the at least one deflecting means and / or the exit end can be changed within the focal plane.

22. Microscope according to one of claims 20 or 21, characterized in that the angle for illuminating light of different wavelength is different.

23. Microscope according to one of claims 12 to 22, characterized in that the microscope has a detector, in particular a multiband detector, which detects synchronously with the switching on of the light source and / or the individual light sources. 24. Microscope according to one of claims 12 to 23, characterized in that the microscope comprises a confocal scanning microscope.

25. Use of a microscope according to one of claims 12 to

24. in biological experiments, in particular in photoactivation and / or caged combound release and / or FRAP (fluorecence recovery after photobleaching).