WO2005033769A1

WO2005033769A1 - Luminescence microscopy

Info

Publication number: WO2005033769A1
Application number: PCT/EP2004/010345
Authority: WO
Inventors: Ulrich Simon; Rainer Danz; Ralf Wolleschensky
Original assignee: Carl Zeiss Jena Gmbh
Priority date: 2003-09-23
Filing date: 2004-09-15
Publication date: 2005-04-14
Also published as: DE10344063A1

Abstract

The invention relates to a multiphoton luminescence microscope comprising at least one excitation radiation-emitting radiation source (3), an optical excitation arrangement (3, 4, 5, 8) that bundles the excitation radiation in a focus (F) onto a specimen (2) to be examined; a detector device (15, 16) for detecting luminescence radiation excited on or in the specimen (2), and; an optical detection arrangement (15, 14, 7, 5), which receives radiation emitted by the specimen (2) and feeds it to the detector device (15, 16). The optical excitation arrangement (3, 4, 5, 8) irradiates the excitation radiation along an axis of incidence (10), which is situated at an angle to a surface normal (11) of the specimen (2), and the optical detection arrangement (12, 14, 7, 5) receives the emitted radiation along a detection axis (13) that is located symmetric to the irradiation axis (10) with regard to the surface normal (11).

Description

Luminescence microscopy

The invention relates to a multi-photon luminescence microscope with at least one radiation source emitting excitation radiation, an optical excitation arrangement that focuses the excitation radiation in a focus on a sample to be examined, a detector device for detecting luminescence radiation excited on or in the sample and an optical arrangement which receives radiation emitted by the sample and supplies it to the detector device. The invention is further directed to a multi-photon luminescence microscopy method in which a sample is illuminated with excitation radiation focused in one focus and radiation emitted by the sample is recorded and detected.

With conventional fluorescence microscopy, luminescence in the sample is excited. A bundled excitation radiation, usually laser radiation, which is matched to the generation of maximum luminescence or fluorescence, is usually used. The excitation takes place in the focus area, luminescence also being stimulated in the incident or emerging light cone of the focused beam. For image generation, the luminescence radiation is recorded by confocal detection only from the area of the focus of the excitation radiation. An image is created by scanning a sample.

In a special form of luminescence microscopy, the sample is excited by a non-linear two- or multi-photon process. The excitation radiation is chosen spectrally so that at least two photons are necessary in order to achieve excitation of the material of the sample causing luminescence radiation. Since the probability of excitation is thus greatly reduced, effective excitation can only take place at very high flux densities, which are only given exactly in the focus of the bundled excitation radiation. Therefore, emission of luminescence or fluorescence radiation is only stimulated at the focal point. This situation is described, for example, in DE 198 01 139 A1, which discloses a microscope or a method of the type mentioned at the beginning. Multi-photon luminescence microscopy requires high photon flux densities to ensure that two photons can work together synergistically. It is therefore an inherent problem of multi-photon luminescence microscopy to achieve the best possible efficiency of the excitation as well as the detection of the luminescence radiation.

It is an object of the present invention to develop a multi-photon luminescence microscope of the type mentioned at the outset or a multi-photon luminescence microscopy method of the type mentioned at the outset in such a way that particularly effective excitation and detection is achieved.

This object is achieved with a multi-photon luminescence microscope of the type mentioned above in that the optical excitation arrangement radiates the excitation radiation along an axis of incidence, which lies at an angle to a surface normal of the sample, and in that the optical detection arrangement detects the radiation emitted in the along a detection axis, which is symmetrical with respect to the surface normal to the radiation axis. The object is further achieved by a multi-photon luminescence microscopy method of the type mentioned at the outset, in which the excitation radiation is irradiated along an axis of incidence which is at an angle to the surface normal of the sample, and the radiation emitted by the sample is recorded along a detection axis which is symmetrical with respect to the radiation axis with respect to the surface normal.

According to the invention, it is therefore provided that the excitation radiation and the emitted radiation are guided along axes which lie symmetrically to a surface normal of the sample. This approach is surprising before the known prior art, which in multi-photon luminescence microscopy assumes that the detector device should record radiation emitted in all spatial directions as far as possible. The invention is based on the finding that, in the case of multi-photon excitation of materials which are optically isotropic per se, as is the case, for example, with silicon bulk material, symmetry breaks can occur in the material. Such defects can occur, for example, with silicon in the form of doping, crystal defects or the effects of surface roughness. The excitation radiation radiated in at an angle to the surface normal and the luminescence radiation detected symmetrically thereto allow such symmetry calculations to be detected particularly sensitively, so that the smaller solid angles from which an external detector as used in the prior art is used to cover the largest possible solid angular range Luminescence radiation is recorded, surprisingly not automatically disadvantageous.

The oblique radiation or detection opens up an additional analysis option if the angle of the radiation is adjusted. In a further development of the multi-photon luminescence microscope, an adjusting device is therefore provided with which the angle of incidence or the detection can be adjusted. The adjustment should preferably take place around a value of 45 °, since in this case a maximum of the luminescence intensity should occur in symmetry breaks in most cases.

A sample is preferably scanned by scanning, i.e. the position of the focus is adjusted on or in the sample. This requires a relative movement between the sample and the microscope, which can be achieved either by a scanning device influencing the optical beam path or alternatively and additionally by a one-dimensional or multi-dimensional translation of the sample.

The concept according to the invention can also be implemented in conventional laser scanning microscopes, as described, for example, in the publication R. Wolleschensky et al., "Characterization and optimization of a laser scanning microscope in the femtosecond regime", Applied Physics B 67, 1998 , Pages 87 - 94. With such a conventional multi-photon luminescence microscope, the oblique radiation of the excitation radiation and the symmetrical oblique detection of the emitted radiation can be realized by a special objective which is inserted into a nosepiece of a conventional laser In such a special objective, the optical excitation arrangement and the optical detection arrangement at least partially have a common beam path.

Such a common beam path enables the sample to be scanned in a simple manner by adjusting the focus. For such a development, it is preferred that the detection arrangement has a first beam splitter, which couples radiation emitted by the sample into the beam path of the excitation arrangement, and a second beam splitter, which couples radiation emitted by the sample out of the beam path of the excitation arrangement. Such a second beam splitter is usually present in conventional laser scanning microscopes. The above-mentioned special objective will therefore have the first beam splitter and optics for focusing the excitation radiation or recording the emitted radiation. If the scanning device lies between the first and second beam splitters, it is ensured in a simple manner that the excitation radiation is focused on a spot from which the emitted radiation is also picked up by means of the detection arrangement.

A particularly simple construction of the special lens mentioned or a particularly expedient realization of the split beam path is a lens with a divided pupil, which is both part of the excitation arrangement and the detection arrangement. Then the excitation radiation can run through one part of the pupil and the detection radiation through the other part. The pupil division is expediently realized by a block filter arranged upstream of the objective with respect to the sample and which filters a part of the pupil of the objective. The filter can, for example, spectrally filter the excitation radiation or the emitted radiation.

To detect the symmetry breaking mentioned at the outset, it is expedient to use linearly polarized excitation radiation, since an additional polarization effect then occurs in such a way that the direction of polarization between the excitation radiation and the detected radiation is shifted by 90 °. It is therefore preferred that the radiation source emits linearly polarized excitation radiation. The beam splitters mentioned at the outset can then expediently be designed as pole splitters. Of course, they can alternatively or additionally be dichroic color dividers.

The method according to the invention makes it possible to detect an intensity maximum of the radiation emitted by the sample by adjusting the angle, and thus to open up an additional analysis possibility.

The sample is preferably scanned at an angle set to the maximum intensity. Alternatively, the sample can also be scanned in a first run at a certain angle, for example equal to or close to 45 °, and the angle can be varied in at least one point of the sample to improve the contrast in a second run.

Alternatively, a selection can also be made as to whether the contrast is above a threshold value within a certain angular range. The contrast dependency can also be evaluated as a function of the angle for image display.

The invention is explained in more detail below by way of example with reference to the drawings. In the drawings: 1 shows a schematic illustration of a laser scanning microscope for multi-photon luminescence microscopy, FIG. 2 shows an example of a special objective of the microscope of FIG. 1, FIG. 3 shows the beam path in the objective of FIG. 2 and FIG Pupil division in the objective of FIG. 2.

A microscope M is shown schematically in FIG. 1, which permits multi-photon fluorescence or luminescence microscopy. The microscope M examines a surface 1 of a sample 2, which is a silicon semiconductor component, for example. A laser 3 emits linearly polarized radiation in the form of short pulses. In one embodiment, the wavelength of the radiation is between 700 nm and 1,000 nm. The radiation is directed via a first beam splitter 4 onto a scanner 5, which enables a two-axis deflection of the light beam in order to scan the surface 1. After the deflection by the scanner 5, the radiation of the laser 3 serving as excitation radiation falls through a tube lens 6, passes through a second beam splitter 7 without being deflected by the latter and is guided into an excitation objective 9 by means of a deflection mirror 8.

The excitation objective 9 focuses the excitation radiation into a focus F on the surface 1 of the sample 2. According to the known principle of laser scanning microscopy, the focus can of course also be in the sample. The focusing takes place in an excitation cone which extends from a lens 9 to the surface 1 along a main axis of the excitation radiation. This main axis 10 lies obliquely at an angle to the surface normal 11 of the surface 1. The short-pulsed laser radiation from the laser 3 causes the generation of fluorescence radiation in or on the sample 2 by non-linear effects. The radiation therefore only arises in focus F.

It is recorded by means of a detection objective, wherein the detection objective 12 is likewise focused on the focus F and thus collects the radiation from the sample 2, a detection cone DK extending along a main axis 13 of the detection. The main axis 13 is symmetrical with respect to the surface normal 11 to the main axis 10.

The received radiation is guided via a deflection mirror 14 to the second beam splitter 7, which is dichroically designed in such a way that it deflects the detected radiation to the tube lens 6 and thus to the scanner 5. The second beam splitter 7, by virtue of its dichroic properties, therefore allows the excitation radiation to pass and reflects the detected luminescent radiation. This passes from the scanner 5 to the first beam splitter 4, which is also dichroic and therefore allows the luminescence radiation to pass, whereas the excitation radiation is reflected. A filter 15 in front of a detector 16 filters the luminescent radiation and blocks any excitation radiation that has entered the beam path to the detector 16. The detector 16 is read out by a control unit 17 which, in addition to controlling the laser 3 and the scanner 5, also controls a scanning table 18 which effects a z-adjustment of the sample 2.

In addition to the filters and optical units described for the microscope 1, additional filters are optionally provided in the region of the deflection mirror 14 or deflection mirror 8, which spectrally condition the excitation radiation in particular or filter excitation radiation from the radiation from the detection cone DK.

The unit consisting of deflecting mirror 8 and excitation objective 9 as well as detection objective 12 and deflecting mirror 14 forms, in one embodiment of microscope M, together with second beam splitter 9 a special objective that can be inserted into a nosepiece of a conventional laser scanning microscope.

In a further embodiment, the special objective is designed as a combination objective 19, which carries both the excitation radiation and the detected radiation. These two radiation components run in beam paths along optical axes that are offset parallel to the optical axis of the combination objective 19. The optical axis of the combination lens 19 coincides with the surface normal 11. The parallel offset of the beam paths ensures that the main axis 10 of the excitation radiation lies obliquely and symmetrically to the main axis 13 of the detection. At the same time, it is ensured that excitation radiation and detection radiation are related to the same focus F.

FIG. 3 shows a possible embodiment of the combination objective 19 in a laser scanning microscope M, only the beam path after the scanner 5 being shown. The combination objective 19 is arranged downstream of the tube lens 6 in the direction of the sample 2. Between the tube lens 6 and the combination objective 19 there is a block filter 20 (in the infinity beam path), which blocks either the excitation radiation or the luminescence radiation to be detected. The combination objective 19 thus has the divided pupil 21 shown in FIG. 4, which includes an excitation part 22 and a detection part 23. This division is effected by the block filter 20.

For microscopy with the microscope M, the angle between the main axis 10 and the surface normal 11 or the angle between the main axis 10 of the excitation radiation and the main axis 13 of the detection can be changed in order to achieve an intensity maximum or an improvement in contrast. This variation can either by suitable adjustment of excitation lens 9 and detection lens 12 or by suitable adjustment of combination lens 19, which can be designed as a zoom lens or with other suitable means.

In a preferred embodiment, the area of surface 1 to be analyzed is scanned in a first pass at an angle of approximately 45 °. A contrast improvement is then achieved at individual points or sub-areas by varying the angle.

Claims

claims

1. Multi-photon luminescence microscope with - at least one radiation source (3) emitting excitation radiation,

- an optical excitation arrangement (3, 4, 5, 8) which focuses the excitation radiation in a focus (F) on a sample (2) to be examined,

- A detector device (15, 16) for detecting luminescence radiation excited on or in the sample (2) and - An optical detection arrangement (15, 14, 7, 5) which receives radiation emitted by the sample (2) and which Feeds detector device (15, 16), characterized in that the optical excitation arrangement (3, 4, 5, 8) radiates the excitation radiation along an incident axis (10) which lies at an angle to a surface normal (11) of the sample (2) and the optical detection arrangement (12, 14, 7, 5) receives the emitted radiation along a detection axis (13) which is symmetrical with respect to the surface normal (11) to the radiation axis (10).

2. Microscope according to claim 1, characterized by an adjusting device by means of which the angle is adjustable, preferably around a value of 45 °.

3. Microscope according to one of the above claims, characterized by a

Scanning device (5) with which the position of the focus (F) on or in the sample (2) can be adjusted, preferably three-dimensionally.

4. Microscope according to one of the above claims, characterized in that the optical excitation arrangement (3, 4, 5, 8) and the optical detection arrangement (12, 14, 7, 5) at least partially a common beam path (7, 6, 5, 4) have.

5. Microscope according to claim 4, characterized in that the detection arrangement comprises a first beam splitter (7), the radiation emitted by the sample (2) in the beam path of the

Coupling excitation arrangement (3, 4, 5, 8), and having a second beam splitter (4), the the sample (2) emits radiation emitted from the beam path of the excitation arrangement (3, 4, 5, 8).

6. Microscope according to claims 3 and 5, characterized in that the scanning device (5) between the first and second beam splitter (7, 4).

7. Microscope according to claim 4, characterized by an objective (19) with a divided pupil (21) which is both part of the excitation arrangement and the detection arrangement.

8. Microscope according to claim 7, characterized by a lens (19) with respect to the sample (2) upstream block filter (2) which filters a part of the pupil (21) of the objective (19).

9. Microscope according to one of the above claims, characterized in that the radiation source (3) emits linearly polarized excitation radiation.

10. Microscope according to claims 5 and 9, characterized in that the first and second beam splitters (7, 4) are designed as pole splitters.

11. Multi-photon luminescence microscopy method in which a sample (2) is illuminated with excitation radiation focused in a focus (F) and radiation emitted by the sample (2) is recorded and detected, characterized in that the excitation radiation is along an axis of incidence (10) is irradiated, which lies at an angle to a surface normal (11) of the sample (2), and the radiation emitted by the sample (2) is recorded along a detection axis (13) which is symmetrical with respect to the surface normal (11) to the radiation axis (10).

12. The method according to claim (11), characterized in that the angle is adjusted in order to detect an intensity maximum of the radiation emitted by the sample (2).

13. The method according to claim 11 or 12, characterized in that the sample (2) is scanned by two- or three-dimensional displacement of the position of the focus (F) on or in the sample (2).

14. The method according to claims 13 and 12, characterized in that the sample (2) is scanned in a first pass with an angle equal to or close to 45 ° and in a second pass at least one point of the sample (2) the angle to Contrast improvement is varied.