WO2005050280A1

WO2005050280A1 - Microscopy device

Info

Publication number: WO2005050280A1
Application number: PCT/EP2004/012495
Authority: WO
Inventors: Albrecht Brandenburg
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2003-11-18
Filing date: 2004-11-04
Publication date: 2005-06-02
Also published as: DE10353694A1; EP1685438A1

Abstract

The invention relates to a microscopy device for observing a sample, and a method for observing a sample in a microscopy device comprising a planar optical waveguide in which a coupled light beam is guided at least in the region of a sample receiving section embodied on the optical waveguide. The inventive device also comprises an objective element oriented towards the sample receiving section.

Description

microscopy apparatus

The present invention relates to a microscope device for observing a sample and a method for observing a sample in a microscope device.

A basic problem of microscopy on three-dimensional objects is that the light signals from different levels of the sample are superimposed. This results in a blurred background that is superimposed on the image from the area of the sharp image. Ideally, one would like the three-dimensional information of the object, i.e. receive the images from different levels in the z direction without influencing the image from other object levels.

Today, three main methods of separating the z-stacks are used. In conventional microscopy, post-processing of the images from different sharpness levels (z-stack) can achieve a subsequent improvement. So-called deconvolution algorithms exist for this. However, methods which achieve the separation of the levels by suitable optical arrangements are generally superior to the post-processing of non-optimal output data.

One such method is laser scan microscopy. The z-stacks are separated by scanning a focused laser beam over the sample. A pin-hole in the imaging beam path ensures that only light from one level reaches the detector. By successively scanning different levels, the complete three-dimensional information is obtained. However, these microscopes are very complex and expensive. Scanning the individual levels also means a relatively high expenditure of time, so that moving objects (e.g. living cells) can only be detected to a limited extent.

Total reflection microscopy (TIRM: Total infernal reflection mnicroscopy) has been used as a further method for a few years. Here, the lighting takes place through the so-called evanescent field, which is created when a light wave is totally reflected on a glass surface (Fig. 1). The evanescent penetrates outside the glass Field with an exponentially declining field or intensity distribution into the adjacent medium. The penetration depth d (1 / e value of the field) depends on the wavelength K, the angle of incidence Φ and the refractive indices of the first (higher refractive) medium ni and the second medium n ₂ : d = λ / [2π (n ₂ ² - ² sin ² Φ) ^1/2 ].

In this way, only the part of the object located directly on the surface is illuminated and accordingly only this part is observed. The penetration depth for a glass - water transition is approx. 200 - 300 nm. This means - similar to laser scanning microscopy - that a cut is made through the sample that is significantly 'thinner ¹ ' than in the case of the laser scan microscope. However, in the case of the TIRM, the position of the observable area is not arbitrary, but is fixed on the surface of the slide.

The TIRM can also be used in combination with fluorescence microscopy. In this case the fluorescence is excited by the evanescent field. The image is recorded in the spectral range of the fluorescence emission. In this case one speaks of TIRFM (Total internal reflection fluorescence microscopy).

Basically, two arrangements of lighting are known; It can be done either through the lens or on the side of the object opposite the lens through a prism. These arrangements have the following advantages and disadvantages: Illumination through the lens has the advantage that the light illuminates the sample lying on the slide without the surrounding medium having to be irradiated. On the other hand, the lens is irradiated. In the case of sensitive fluorescence-optical detection methods, the scattered light which arises in the lens can considerably disrupt the measurement, since the excitation intensities are orders of magnitude higher than the light intensities to be detected. Even with good interference filters, so much excitation light can get into the detection unit that a disturbing background is found. The alternative method is excitation on the side of the object opposite the lens. Since the total reflection condition is not achieved by irradiation on one side of a plane-parallel plate, a prism must be used here. In this case, the excitation and emission beam paths are separated. However, in this case the sample space must be over the area to be observed on the slide surface are irradiated. As a rule, there are biological samples, for example in buffer media. Reagents or nutrients may be added to these media during observation. However, this can severely disturb the observation beam path, which among other things reduces the achievable resolution. The part of the sample that is at a greater distance from the prism surface can also contribute to the reduction of the image quality by absorption or scattering of the light.

It is the object of the present invention to provide a microscopy device for observing a sample and a method for observing a sample in a microscopy device, wherein an observation of the sample with high image quality is possible.

With regard to the device aspect, this object is achieved according to the invention by a microscopy device for observing a sample with: a planar optical waveguide on which a sample receiving section is provided for receiving the sample, a light coupling device for coupling light into the planar optical waveguide, the planar optical waveguide for guidance of a coupled-in light beam is provided at least in the region of the sample receiving section, and an objective device which is directed onto the sample receiving section.

The sample receiving section is preferably arranged directly on the planar optical waveguide and extends essentially parallel to the planar optical waveguide.

The objective device is preferably directed through the planar optical waveguide onto the sample receiving section.

According to a preferred embodiment, the planar optical waveguide is arranged on a carrier substrate. According to a preferred embodiment, the carrier substrate consists of a transparent carrier material. The objective device is preferably directed through the transparent carrier substrate onto the sample receiving section.

According to a preferred exemplary embodiment, a coupling section is provided for the direct coupling of the light beam onto the planar optical waveguide.

According to a further preferred exemplary embodiment, a coupling section is provided for coupling the light beam into the planar optical waveguide on the transparent carrier substrate.

The coupling section is preferably provided for coupling the light beam outside the sample receiving section.

According to a preferred embodiment, the coupling section has a coupling grating.

According to a further preferred exemplary embodiment, the coupling section has a coupling prism.

The planar optical waveguide is preferably designed as a coating on the carrier substrate. According to a preferred embodiment, the coating is made of a tantalum oxide layer, in particular a tantalum pentoxide layer (Ta ₂ Os), or a silicon nitride layer (Si ₃ N) or a silicon oxynitride layer (Si _x O _y N _z ) or a titanium oxide -Layer (TiO ₂ ) formed on the carrier substrate 5 made of glass or polymer.

The sample receiving section is preferably covered by a cover substrate.

According to a further preferred exemplary embodiment, the objective device is directed onto the sample receiving section essentially perpendicular to the guided light beam. With regard to the method aspect, this object is achieved according to the invention by methods for observing a sample in a microscopy device, the sample being arranged on a sample receiving section of a planar optical waveguide, and an injected light beam being guided in the planar optical waveguide at least in the region of the sample receiving section, and that Sample is observed through a lens device.

The direction of observation is preferably substantially perpendicular to the guided light beam.

The sample is preferably applied directly to the planar optical waveguide and the observed part of the sample extends substantially parallel to the guided light beam.

The sample is preferably observed through the planar optical waveguide.

The planar optical waveguide is preferably arranged on a transparent carrier substrate, and the sample is observed through the transparent carrier substrate.

According to a preferred embodiment, the light beam is coupled directly into the planar optical waveguide.

According to a further preferred exemplary embodiment, the light beam is coupled into the planar optical waveguide via the transparent carrier substrate.

The light beam is preferably coupled in outside the sample receiving section.

An apparatus and a method are thus advantageously created, whereby on the one hand a separation between the excitation and observation beam path is ensured, but at the same time manages without the passage of the light through the sample space. In addition, the depth of penetration is further reduced by Advantage. In addition, a high excitation intensity on the surface is required in particular in the case of fluorescence analyzes. The passage through other media (including the slide) should contribute as little as possible to the background light. This disturbing light is created both by scattering and by the natural fluorescence of the media used.

The present invention is described in more detail below using exemplary embodiments in conjunction with the accompanying drawings. The drawings show:

1 shows an embodiment of the microscopy device in a schematic representation,

Fig. 2 shows the intensity distribution in an optical waveguide on a carrier substrate according to an embodiment.

1 shows a schematic representation of a microscopy device according to an exemplary embodiment. This microscopy device has a planar optical waveguide 1. A sample receiving section 2 is provided on this planar optical waveguide 1, which is essentially designed as a thin, flat plate. This sample receiving section 2 is arranged directly on a side surface of the planar optical waveguide 1.

The sample or substance to be examined is thus placed on the side surface of the planar optical waveguide, so that this contact plane is the observation or. Analysis level of the sample determined. In the exemplary embodiment shown, the sample receiving section 2 and the sample space determined thereby are defined by spacer elements 8, a cover substrate 7 in the form of a cover glass delimiting the sample space at the top.

In the exemplary embodiment shown, a coupling section 6 is provided on the optical waveguide 1 outside of the sample receiving section 2 in order to couple light L, which is emitted by a light source (not specified in any more detail) into the planar optical waveguide 1. The coupled light beam 3 extends, as shown in FIG. 1, along the planar optical waveguide 1, starting from the coupling section 6, along the sample receiving section 2. Thus, the coupled light beam extends essentially parallel to the sample receiving section 2, ie essentially parallel to the contact plane of an applied sample with the optical waveguide 1. It follows that the coupled light beam also extends essentially parallel to the observation plane.

This coupled light beam 3 in the planar optical waveguide 1 generates an evanescent field that extends essentially perpendicular to the coupled light beam 3. The evanescent field emerges from the optical waveguide 3 so that the edge regions along the optical waveguide 1 are illuminated. This illumination of the edge regions with a very shallow depth takes place essentially perpendicular to the light beam 3 that is coupled in.

In the exemplary embodiment shown, the evanescent field of the coupled-in light beam 3 enters the sample receiving section 2 and thus illuminates the observation plane, i.e. the contact plane of the sample to be observed on the optical waveguide 1. This contact plane of the sample can be observed via the objective device 4. The lens device 4 is directed through the optical waveguide 1 onto the sample receiving section 2. In the exemplary embodiment shown, the direction of observation of the objective device 4 is essentially perpendicular to the planar optical waveguide 1 and thus essentially perpendicular to the coupled light beam 3.

In the exemplary embodiment shown, the planar optical waveguide 1 is arranged on a carrier substrate 5. This carrier substrate 5 is made of a transparent material, such as glass, so that the objective device 4 can observe the observation plane of the sample through the optical waveguide 1 and through the transparent carrier substrate 5.

In the microscopy device shown in FIG. 1, excitation takes place through the planar optical waveguide 1. The evanescent field of this optical waveguide penetrates into the a sample lying in the area of the sample receiving section 2.

The optical waveguide can consist, for example, of a thin plate made of a transparent material. This thin plate consists for example of a glass (e.g. slide or cover glass format), a polymer or a transparent crystalline material. The thickness of this thin plate is in the range 0.1-2 mm. The length of this thin plate is in the range of 1.0 - 5.0 cm, the width 0.5 - 5.0 cm. The refractive index of this thin plate is greater than the refractive index of the surrounding media for the formation of the light guide. For example, the refractive indices of the materials mentioned are in the range 1.4-1.6.

The optical waveguide 1 of the exemplary embodiment shown consists of a thin coating which has been applied, for example, to a glass slide (for example slide or cover glass format), a polymer or a transparent crystalline material (support substrate 5). In addition to the tantalum pentoxide (Ta ₂ O ₅ ) described below, the coating can also consist of silicon nitride (Si ₃ N), silicon oxynitride (Si _x O _y N _z ) or titanium oxide (TiO ₂ ). In any case, the refractive index of the coating material must be greater than the refractive index of the carrier material and greater than that of the surrounding medium.

Ideally, the difference between the refractive indices of the support and the coating should be as large as possible, since in this case the evanescent field portion is particularly large. This makes the arrangement sensitive to changes on the waveguide surface. The coating materials mentioned are therefore preferably high-index coatings. In addition to the refractive index, the chemical resistance and low light scatter are important selection criteria for the selection of the coating. The thickness of the waveguiding coating is advantageously chosen so that only the basic mode of the waveguide is capable of propagation. Only in this case is the intensity distribution in the waveguide and the evanescent field clearly defined. In addition, the basic mode has the highest evanescent field with an optimized waveguide thickness. Typical thicknesses are in the range 120 nm - 500 nm. The substrate refractive indices are in the range 1, 4 -1, 6. The refractive indices of the coatings are typically 1.55-2.4. The coupling grid is either in the surface of the carrier (before coating with the waveguide material) or etched into the coating. The grating periods are typically in the range 300 nm - 1000 nm. The etching depths are in the range 5 nm - 50 nm.

As shown in Fig. 1, the excitation from the sample side takes place with separate beam paths. In the case of the optical waveguide 1 consisting of a coating, the excitation light scarcely shines through the glass (carrier substrate 5) (apart from the evanescent field on the substrate side), so that any intrusive fluorescence of the glass is reduced. The observation is carried out through the glass support (support substrate 5) without radiating through the sample space (sample receiving section 2).

In the simplest case, the coupling takes place in the case of the glass carrier forming the optical waveguide through an obliquely polished edge. In the case of the optical waveguide consisting of a thin coating, e.g. a coupling grid (coupling section 6) is used, which is introduced into the carrier by etching or impression techniques. Alternatively, the coupling can take place by means of a coupling prism applied to the optical waveguide. The light can be coupled into the grating both from the coating side and from the carrier side into the optical waveguide.

Another advantage of very thin, highly refractive optical fibers is that the intensity of the evanescent field is very high and the extent into the adjacent medium can be limited to approx. 50 nm, so that the observation volume is limited very closely. Signals from other levels in the sample are suppressed.

A tantalum oxide layer on glass is given as an example of a coating waveguide. With the waveguide data

Film refractive index nf = 2.22 (Ta ₂ O ₅ )

• Refractive index n _s = 1,515 (glass)

• ambient refractive index n _u = 1.33 (water)

• Film thickness d = 156 nm

• Light wavelength λ ₀ = 633 nm arithmetically for the evenescent field:

• for the basic mode (m = 0) in TE polarization: n _eff = 1, 92530

• Intensity on WL surface l (0) / l _max = 0.39

• Half-width of the evanescent field x _{1 2} = 25 nm

• Power P _u / P = 0.18 carried in the surrounding medium

l (0) denotes the intensity at the waveguide surface,

Imax the maximum intensity of the distribution in the waveguide,

Half-width of the evanescent field denotes the distance from the waveguide surface (outside the waveguide) at which the intensity assumes half the intasity on the surface of the waveguide.

Power in the surrounding medium Pu denotes the light power that is conducted on the surface outside the waveguide, P is the total intensity of the intensity distribution.

The intensity distribution of such an optical waveguide with a carrier substrate is shown in FIG. 2.

The exemplary embodiment described above shows a microscopy device for observing a sample with a planar optical waveguide 1, on which a sample receiving section 2 is provided for receiving the sample. A light coupling device for coupling light into the planar optical waveguide 1 is provided, the planar optical waveguide 1 for guiding a coupled light beam 3 at least in the area of the sample receiving section 2. A lens device 4 is directed onto the sample receiving section 2.

The sample receiving section 2 is arranged directly on the planar optical waveguide 1. The sample receiving section 2 extends essentially parallel to the planar optical waveguide 1. The objective device 4 is directed through the planar optical waveguide 1 onto the sample receiving section 2. The objective device 4 is directed essentially perpendicular to the guided light beam 3 onto the sample receiving section 2.

According to the exemplary embodiment shown, the planar optical waveguide 1 is arranged on a carrier substrate 5. This carrier substrate 5 consists of a transparent carrier material. The objective device 4 is directed through the transparent carrier substrate 5 onto the sample receiving section 2.

In the exemplary embodiment shown, a coupling section 6 is provided for directly coupling the light beam 3 onto the planar optical waveguide 1. The coupling section 6 is provided for coupling the light beam 3 outside the sample receiving section 2. The coupling section 6 has a coupling grid. As an alternative to the exemplary embodiment shown, a coupling section can be provided on the transparent carrier substrate for coupling the light beam into the planar optical waveguide. As an alternative to the coupling grating shown, the coupling section can have a coupling prism.

In the exemplary embodiment shown, the planar optical waveguide 1 is designed as a coating on the carrier substrate 5. The coating is made of a tantalum oxide layer, in particular a tantalum pentoxide layer (Ta ₂ O ₅ ), or a silicon nitride layer (Si ₃ N) or a silicon oxynitride layer (Si _x O _y N _z ) or a titanium oxide layer ( TiO ₂ ) formed on the carrier substrate 5 made of glass or polymer. In the exemplary embodiment shown, the sample receiving section 2 is covered by a cover substrate 7 made of glass.

The exemplary embodiment described above also shows a method for observing a sample in a microscopy device. The sample is arranged on a sample receiving section 2 of a planar optical waveguide 1. A coupled light beam 3 is guided in the planar optical waveguide 1 at least in the area of the sample receiving section 2. The sample is observed through an objective device 4. The direction of observation is essentially perpendicular to the guided light beam 3. The sample is observed through the planar optical waveguide 1.

The sample is applied directly to the planar optical waveguide 1, and the observed part of the sample extends essentially parallel to the guided light beam 3. In the exemplary embodiment shown, the planar optical waveguide 1 is arranged on a transparent carrier substrate (5), and the sample is observed through the transparent carrier substrate (5).

In the exemplary embodiment shown, the light beam 3 is coupled directly into the planar optical waveguide 1. As an alternative to the exemplary embodiment shown, the light beam can be coupled into the planar optical waveguide via the transparent carrier substrate.

In the exemplary embodiment shown, the light beam 3 is coupled in outside the sample receiving section 2.

Claims

claims:

1. Microscopy device for observing a sample, comprising: a planar optical waveguide (1) on which a sample receiving section (2) is provided for receiving the sample, a light coupling device for coupling light into the planar optical waveguide (1), the planar optical waveguide (1 ) for guiding a coupled light beam (3) at least in the area of the sample receiving section (2), and an objective device (4) which is directed onto the sample receiving section (2).

2. Microscopy device according to claim 1, characterized in that the sample receiving section (2) is arranged directly on the planar optical waveguide (1) and extends substantially parallel to the planar optical waveguide (1).

3. Microscopy device according to claim 1 or 2, characterized in that the objective device (4) through the planar optical waveguide (1) is directed to the sample receiving section (2).

4. Microscopy device according to at least one of claims 1 to 3, characterized in that the planar optical waveguide (1) is arranged on a carrier substrate (5).

5. Microscopy device according to claim 4, characterized in that the carrier substrate (5) consists of a transparent carrier material.

6. Microscopy device according to claim 5, characterized in that the objective device (4) through the transparent carrier substrate (5) is directed to the sample receiving section (2).

7. Microscopy device according to at least one of claims 1 to 6, characterized in that a coupling section (6) for the direct coupling of the light beam (3) on the planar optical waveguide (1) is provided.

8. Microscopy device according to at least one of claims 4 to 6, characterized in that a coupling section is provided for coupling the light beam into the planar optical waveguide on the transparent carrier substrate.

9. Microscopy device according to at least one of claims 7 or 8, characterized in that the coupling section (6) for coupling the light beam (3) is provided outside the sample receiving section (2).

10. Microscopy device according to at least one of claims 7 to 9, characterized in that the coupling section (6) has a coupling grating.

11. Microscopy device according to at least one of claims 7 to 9, characterized in that the coupling section has a coupling prism.

12. Microscopy device according to at least one of claims 4 to 11, characterized in that the planar optical waveguide (1) is designed as a coating on the carrier substrate (5).

13. Microscopy device according to claim 12, characterized in that the coating of a tantalum oxide layer, in particular a tantalum pentoxide layer (Ta ₂ Os), or a silicon nitride layer (Si ₃ N ₄ ) or a silicon oxynitride layer (Si _x O _y N _z ) or a titanium oxide layer (TiO ₂ ) is formed on the carrier substrate (5) made of glass or polymer.

14. Microscopy device according to at least one of claims 1 to 13, characterized in that the sample receiving section (2) is covered by a cover substrate (7).

15. Microscopy device according to at least one of claims 1 to 14, characterized in that the objective device (4) is directed substantially perpendicular to the guided light beam (3) on the sample receiving section (2).

16. A method for observing a sample in a microscopy device, the sample being arranged on a sample receiving section (2) of a planar optical waveguide (1), and an injected light beam (3) in the planar optical waveguide (1) at least in the region of the sample receiving section (2 ) and the sample is observed through an objective device (4).

17. A method for observing a sample in a microscopy device according to claim 16, characterized in that the direction of observation is substantially perpendicular to the guided light beam (3).

18. A method for observing a sample in a microscopy device according to claim 16 or 17, characterized in that the sample is applied directly to the planar optical waveguide (1), and the observed part of the sample is substantially parallel to the guided light beam (3) extends.

19. A method for observing a sample in a microscope device according to at least one of claims 16 to 18, characterized in that the sample is observed through the planar optical waveguide (1).

20. A method for observing a sample in a microscopy device according to at least one of claims 16 to 19, characterized in that the planar optical waveguide (1) is arranged on a transparent carrier substrate (5), and the sample is observed through the transparent carrier substrate (5) becomes.

21. A method for observing a sample in a microscopy device according to at least one of claims 16 to 20, characterized in that the light beam (3) is coupled directly into the planar optical waveguide (1).

22. A method for observing a sample in a microscopy device according to claim 20, characterized in that the light beam is coupled into the planar optical waveguide via the transparent carrier substrate.

23. A method for observing a sample in a microscopy device according to at least one of claims 16 to 23, characterized in that the light beam (3) is coupled in outside the sample receiving section (2).