WO2009043471A1

WO2009043471A1 - Arrangement for the optical detection of light radiation excited in and/or scattered back from a sample

Info

Publication number: WO2009043471A1
Application number: PCT/EP2008/007685
Authority: WO
Inventors: Ralf Wolleschensky
Original assignee: Carl Zeiss Microimaging Gmbh
Priority date: 2007-09-28
Filing date: 2008-09-16
Publication date: 2009-04-09
Also published as: DE102007047467A1

Abstract

The invention relates to an arrangement for the optical detection of light radiation excited in and/or scattered back from a sample, wherein the sample illumination has a greater chronological coherence length than the detection radiation, and wherein a substantially cascaded step element having step heights that are greater than the coherence length of the detection light, and less than that of the excitement light is provided, wherein a partial beam of the light is generated at each step, and a change of beam properties of the excitement light occurs by interference of the partial beams of the excitement light, or wherein the probe illumination has a shorter chronological coherence length than the detection radiation, and a substantially cascading step element is provided, having step heights shorter than the coherence length of the detection light, and greater than the excitement light, wherein a partial beam of the light is generated at each step, and a change of the beam properties of the detection light occurs by interference of the partial beams of the detection light.

Description

Title:

Arrangement for the optical detection of light radiation excited and / or backscattered in a sample

The invention relates to an achromatic main color splitter for laser scanning microscopy, which allows efficient excitation and detection in preferential confocal imaging and the integration thereof into a microscope, in particular a laser scanning microscope.

State of the art

In a prior art laser scanning microscope (FIG. 1), light from the light source (LQ) passes through a beam splitter (MDB), the scanners (SC), the scanning optics (SO), the tube lens (TL), and the Objective (O) punctiform on the sample (PR). In the sample, for example, fluorescent light is excited, which is collected by the lens and in turn reaches the beam splitter. The beam splitter is designed such that it transmits the fluorescent light due to the spectral properties which are changed with respect to the excitation wavelength, so that the detection light is focused with a pinhole optic (PO) through a confocal diaphragm (PH) and subsequently reaches a detector (DE). In front of the detector, an emission filter (EF) for suppressing the excitation light is provided in a fluorescence microscope. With the help of the scanner, the illumination spot is scanned over the sample. The sample signals are combined in a computer to form an image.

Such beam splitters are described for example in DE 19702753 A1. From the beach of technology, the use of the polarization state of the light is known for beam splitting (MDB). For this purpose, the excitation radiation is linearly polarized. However, the fluorescence is usually unpolarized. By forming the MDB as polarization splitter, however, only 50% of the fluorescence radiation can be separated from the excitation radiation achromatically.

Furthermore, from the beach of the art a pupil division for the realization of the beam splitting is known (DE 10257237 A1). For this purpose, the excitation light is imaged into the sample with a reduced numerical aperture. Since the fluorescence in all spatial directions is radiated, the collection of fluorescence takes place here in an enlarged aperture.

For fluorescence excitation a wide variety of dyes are used. This means that for different excitation and detection wavelengths different color splitters are needed, which can normally be swung in and out of the beam path on a filter wheel. In addition, preparations with several dyes are provided simultaneously. The recording of the sample signals, so-called multiple fluorescence signals, then takes place with a plurality of detectors.

Further details of the prior art on beam splitters in laser scanning microscopes can be found in "Handbook of biological confocal microscopy", Chapter 9, Editor J. P. Pawley, Plenum Press, 1995.

The object of the invention is to achieve an achromatic beam separation of the excitation light from the detection light with high efficiency both for the excitation light and for the detection light. This is realized by the arrangements explained below.

Description of the invention

FIGS. 1-6 and 9-11 each describe arrangements for separating the common excitation and detection beam path. The basic structure of the beam splitting is shown schematically in Fig. 1 based on the prior art. The excitation beam path here comprises beam paths (3) from the direction of the light source (LQ), which are reflected by the beam splitter (MDB) in the direction of the sample (PR). The beam path from the beam splitter (MDB) to the sample (PR) is referred to as a common excitation and detection beam path (1). For example, fluorescent light (FL) is excited in the sample. In addition, the sample reflects a portion of the excitation light (AL). The fluorescent light is preferably transmitted by the beam splitter (MDB) and enters the detection beam path (2), which includes the beam paths from MDB to the detector (DE). Excitation light reflected by the sample (PR) predominantly passes through the MBD in the direction of the excitation beam path (3) and thus in the direction of the light source (LQ). In a laser scanning microscope, the detection light (for example

Fluorescent light) has a larger spectral bandwidth than the excitation light. This is because the excitation occurs in bands of approximately up to 20 nm spectral width. The spectral bandwidth influences the temporal coherence length of the light.

The wider the spectrum of the light, the shorter the temporal coherence length of the light. Fluorescent light thus has a shorter coherence length than that

Excitation light.

According to the invention, it has been recognized that by utilizing this property, a particularly efficient and achromatic beam splitting can be realized.

The beam splitter according to the invention advantageously does not change the imaging properties as compared to the prior art, or only insignificantly.

Fig. 2a) shows an optically transparent step element (PM). This element is characterized in that it has along the optical axis (o.A.) steps with a width (B) and a height (A). The steps can, for example, by successive set glass plates with a lateral offset, by etching processes

(diffractive optics) or realized by micromaterial processing.

The height (A) is chosen so that it is greater than the temporal

Coherence length of the light to be detected. However, the height (A) is smaller than that

Coherence length of the excitation light. For example, light having a fluorescence in the range of 500 to 550 nm has a coherence length of 3.6 μm. The inspiring

Laser light, in contrast, has a narrow band depending on the laser medium

Coherence length in the cm range. The step height could therefore preferably be greater than about 5-10 microns.

The width (B) of the step element (PM) is chosen so that it is greater than the spatial coherence length, so that the step element is not light-bending for the

Excitation and fluorescent light acts.

Fig. 2b) shows the behavior of the element explained in Fig. 2a) in the beam path. Detection and excitation light (1) reach along the optical axis oA on the step element (PM), which is perpendicular (the end face SF of Figure 2a) is arranged to the optical axis in the beam path. The light is split into sub-beams (TrT _n ) at the stages, with adjacent sub-beams giving a discrete phase shift to the height of stage (A) to each other. Since the height (A) is greater than the coherence length of the detection light for the detection light, the partial beams generated at the stages can no longer interfere. Thus, the partial beams of the detection light FL pass without angle change (2) through the step element.

However, the excitation light AL has a longer coherence length than the step height (A). Thus, the partial beams can interfere with each other. This results in a deflection of the excitation light out of the optical axis according to the well-known Snell's law. Here, above all, the first diffraction order is considered. By appropriate design of the step element, for example via the dimensioning of the step width (B) or a slight rotation of the step element to the optical axis, the zeroth order or higher orders can be largely avoided.

This results in a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light.

Fig. 3a) shows a further behavior of the explained with reference to Fig. 2a) element in the beam path. Detection and excitation light (1) illuminate the step element (PM) in a circular manner along the optical axis (O in FIG. 3b), wherein the step element is again arranged perpendicular to the optical axis in the beam path. The light is decomposed at the stages into sub-beams (Ti-T _n ) (FIG. 3 c), with adjacent sub-beams giving a discrete phase shift to the height of stage (A) relative to each other (pupil P). Downstream of the step element is a lens (L).

Since the height (A) is greater than the coherence length of the fluorescent light for the fluorescent light, the partial beams generated at the stages can no longer interfere. Thus, the partial beams of the fluorescent light FL pass without angle change (3) through the step element. In addition, each sub-beam is imaged individually and independently of the other sub-beams through the lens. Decisive for the imaging by the lens is the extension of the partial beams, ie the width of the step (B) and the beam diameter. Since the beam diameter is larger than the width (B), a line-shaped distribution along the x-axis (a in FIG. 3d) arises in the focus of the lens (image) for the fluorescent light. Since the excitation light AL has a greater coherence length than the fluorescent light, it experiences a deflection in the x direction (FIG. 3). The lens continues to focus in both axes, resulting in a point focus (b in Figure 3d). This in turn creates a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light. Through the use of the lens and thus by focusing the excitation light, a simplified spatial separation of excitation and detection light is made possible.

Fig. 4a) shows a further arrangement with which the coherence properties of the excitation and detection light can be used for efficient beam separation. The step element PM in this case has a rotationally symmetrical structure about the optical axis (o. A.) On. In addition, the envelope of the step structure (a) preferably describes a convex lens shape. The step height is again larger for the detection light and smaller for the excitation light than the coherence length of the radiation. Due to the structure, light is split into annular partial beams with the same phase. For the detection light, these partial beams do not interfere. Thereby, the beam shape and direction of the detection light FL is not affected. For the excitation light, the partial beams interfere and produce a focal point at the focal point of the phase element PM. The focal point results from the convex shape of the envelope of the step element. The figure shows a simplified representation of the lens with a straight end surface. This surface can be shaped accordingly. At the focal point, a spatial separation of the excitation and detection light can take place with a small mirror R in the otherwise transmissive beam splitter (MDB), the excitation light AL being reflected in the direction of the output (3). The shape of the MDB is illustrated in part 4b). It has a mirrored region (R) and a transparent region (T). This in turn creates a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light.

Fig. 5) shows an arrangement with which the detection light can be refocused after separation from the excitation light. For this purpose, a compensation element (cPM) is arranged after the step element (PM). The cPM is such that the modified phase front introduced by PM is compensated again. For this, the cPM must have exactly the inverted (mirror-inverted) form of the PM. Substituting both elements PM and cPM together, then results in a plane-parallel plate.

Fig. 6) shows an advantageous combination of the arrangements of Figs. 4 and 2b. Fig. 4 uses a special mirror (MBD) for the spatial splitting of the excitation (3) and detection light (2), whereby the MDB shadows a part of the detection light so that efficiency losses occur. Due to the combination of the deflecting (PM1) and a focusing (PM2) step element, the focusing of only the excitation light outside the detection beam path takes place on a reflector M, so that a particularly efficient beam separation is made possible.

Analogously to FIG. 5, compensation elements cPM1 and cPM2 are provided in the further beam path of the fluorescent light, cPM1 for compensation of PM2 is designed as hollow element HS with cavities H mirrored to PM2, otherwise all formed as a transmissive element, while cPM2 is mirror-inverted to PM1. The phase offset of the elements PM1, PM2 is hereby canceled and at the output of cPM2 fully focusable detection light FL is present.

The step elements (PM) can also be designed to be reflective without restriction. An example of a step element is shown in Fig. 7).

The common excitation and detection light is directed from direction (1) onto the step element. At the step element partial beams of different phases are generated. The step difference (h) is chosen so that the coherence length of the fluorescent light is smaller than the step difference. The detection light (2) thereby experiences a classical reflection, the incidence (a1) and the angle of departure (a2) being equal, since the partial beams (determined by the width of the steps (b)) can not interfere. However, the step difference (h) is smaller than the coherence length of the excitation light. Thus, the sub-beams of the excitation light can not interfere. By interfering with the partial beams of the excitation light (2), however, this undergoes a diffraction on a grid, wherein the Failure angle (a3), then by the number of strips (step width b) according to the lattice formula known from the prior art

a3 = a1 + lambda / b

where lambda is the wavelength of the excitation light. A spatial separation of the excitation from the detection light (angle between the beam paths (2) and (3) = a3-a2) can be done by a suitable choice of the number of stripes (step width b) or the angle of incidence on (PM) (a1). The use of reflective devices has the additional advantage of being achromatic, at least for the detection light.

Fig. 8) shows two exemplary schematic arrangements in a laser scanning microscope, wherein a spatial separation of the beam paths for the excitation and detection light due to the different coherence properties of the light is realized. Here, the excitation light of the light source is coupled in the same direction via the step element as it is decoupled after returning from the sample. Partial image a) shows an arrangement according to the Fig. 1, wherein the beam splitter element (MDB) has been replaced by a beam splitter (S) according to the invention. In the detection beam path (2), the beam splitter (S) is designed so that the phase front modified by the step element (PM) is again smoothed by the element cPM. As a result, the light can be focused on the output side through the confocal diaphragm (PH).

Partial image b) shows a further arrangement, wherein the confocal diaphragm (PH) is arranged in the common excitation and detection beam path (1). Compensation of the phase front modified by the step element (PM) is not required in the detection beam path (2). This has the advantage that the adjustment is simplified because no compensating element (cPM) is used.

The compensation element CPM shown in partial image a) can also be dispensed with if a rectangular diaphragm is used as the confocal diaphragm PH. For the beam splitter preferably an arrangement according to Fig. 2b) is used. The aspect ratio of the length to the width of the rectangle will be preferably chosen such that it corresponds to the ratio of the width of the steps of the step element (B) to the beam diameter at the entrance of the step element.

Fig. 9) shows an example of a detailed representation of the arrangement according to Fig. 8a) with the beam splitter element of Fig. 6).

The beam splitting, i. the beam paths for the excitation and detection light are identical for the outward and return paths. Thus, it does not matter, for example, for the excitation light, whether an irradiation of the light source LQ or a decoupling of the excitation light (which was reflected at the sample) takes place in the direction of the light source - see also explanation above, in particular to FIG. 8a.

Fig. 10) shows an example of a detailed representation of the arrangement of Fig. 8a) with the beam splitter element of Fig. 5).

Fig. 11) shows an example of a detailed representation of the arrangement for a light microscope with a CCD detector, wherein the beam splitter element of Fig. 5) is used for beam splitting. Plotted drawn is the pupil beam, pulled through the object beam path.

In describing the arrangements, it has been assumed that the detection light has a shorter coherence length than the excitation light. If excitation light with a shorter coherence length compared to the detection light is used (white light source), then the step element is designed so that the step height is smaller than the coherence length of the detection light but greater than that of the excitation light. This means that with respect to the arrangements described, the excitation light would pass through the step element and the detection light (fluorescence) would be interfered and diffracted out of the optical axis. In the described arrangements, only the excitation and detection beam path is then interchanged.

Claims

claims

1.

Arrangement for the optical detection of light radiation excited and / or backscattered in a sample, wherein the sample illumination has a greater temporal

Has coherence length as the detection radiation, characterized in that a substantially step-shaped step element with step heights greater than that

Coherence length of the detection light and is smaller than that of the excitation light is provided, wherein at each stage a partial beam of the light is generated and by interference of the partial beams of the excitation light, a change in the

Beam properties of the excitation light takes place.

Second

Arrangement for optical detection of light radiation excited and / or backscattered in a sample, wherein the sample illumination has a smaller temporal

Has coherence length as the detection radiation, characterized in that a substantially step-shaped step element with step heights smaller than that

Coherence length of the detection light and is greater than that of the excitation light is provided, wherein at each stage, a partial beam of the light is generated and by interference of the partial beams of the detection light, a change in the

Beam properties of the detection light takes place.

Third

Arrangement according to one of the preceding claims, wherein the step height is the difference of the optically effective length in the step element between two adjacent stages.

4th

Arrangement according to one of the preceding claims, wherein the change of the beam characteristics in a change of direction of

Interference light with respect to the direction of non-interfering light.

5th

Arrangement according to one of the preceding claims, wherein the step element in the direction of the optical axis of the excitation and / or detection light in its cross section has a step-like decreasing thickness.

6th

Arrangement according to one of the preceding claims, wherein a step element, which deflects the excitation light relative to the detection light, is provided.

7th

Arrangement according to one of the preceding claims, wherein a step element, which focuses the excitation light relative to the detection light in at least one axis, is provided.

8th.

Arrangement according to one of the preceding claims, wherein an additional element for the spatial separation of the excitation and detection beam path is provided.

9th

Arrangement according to one of the preceding claims, wherein the step element is reflective and / or transparent.

10th

Arrangement according to one of the preceding claims, wherein a confocal rectangular aperture is provided at the entrance of the step element.

11th

Arrangement according to one of the preceding claims, wherein a second step element for compensating the phase front is provided in the detection beam path.

12th

Arrangement according to one of the preceding claims, wherein

The formation of the second step element with respect to the irradiation direction for

Training the first step element is substantially mirror inverted.

13th

Arrangement according to one of the preceding claims, wherein an optics for separate focusing of the excitation and the fluorescent light is provided.

14th

Arrangement according to one of the preceding claims, wherein the step element has a rotationally symmetrical structure about the optical axis and an envelope of the step structure preferably has a convex

Lentil shape has.

15th

Arrangement according to claim 14, wherein a second step element is provided which has a cavity which the

Form of the rotationally symmetric step element substantially corresponds.

16th

Arrangement according to one of claims 14,15, wherein the second step element is arranged mirror-inverted to the first step element.

17th

Arrangement according to one of the preceding claims, wherein the excitation light is focused outside of the detection beam path to a mirror.

18th

Arrangement according to one of the preceding claims, wherein via one of the excitation light via a mirror in the illumination beam path and / or is coupled out.

19th

Application of an arrangement according to one of the preceding claims in a microscope.

20th

Application of an arrangement according to one of the preceding claims, in one

Laser scanning microscope.

21st

Microscope, in particular laser scanning microscope according to one of the preceding

Claims.