DE10118463A1 - Depth-resolved optical imaging method for use in biological scanning microscopy, uses phase or frequency modulation of the imaging light - Google Patents

Abstract

Sample is scanned with light of at least one wavelength with detection of the light based on reflection, fluorescence, luminescence, transmission or scattering of the incident light. The illuminating light is modulated in at least one spatial direction. The scanning motion and the detection is achieved using two different phase modulation angles or modulation frequencies, so that a resultant section can be calculated through the sample. Independent claims are also included for: (1) An arrangement for depth-resolved optical imaging of a sample; (2) A method for parallel excitation of a sample interaction.

Description

The invention relates to a method and an arrangement in the Microscopy, especially fluorescence laser scanning microscopy Examination of predominantly biological samples, preparations and associated components. Are included on Fluorescence detection based methods for screening active substances (High Throughput Sceening) as well as on other contrast mechanisms based methods of laser scanning microscopy.

State of the art

A classic application of light microscopy for examination of biological preparations is fluorescence microscopy (Lit .: Pawley, "Handbook of Biological Confocal Microscopy"; Plenum Press 1995). in this connection are certain dyes for the specific marking of cell parts used.

The irradiated photons of a certain energy excite the dye molecules by absorbing a photon from the ground state to an excited state. This excitation is usually referred to as single-photon absorption ( Fig. 1a). The dye molecules excited in this way can return to the ground state in various ways. In fluorescence microscopy, the transition with the emission of a fluorescence photon is most important. The wavelength of the emitted photon is generally red due to the Stokes shift compared to the excitation radiation, so it has a longer wavelength. The Stokes shift enables the fluorescence radiation to be separated from the excitation radiation.

The fluorescent light is in with suitable dichroic beam splitters Combination with block filters split off from the excitation radiation and observed separately. This is the representation of individual, with different Colored cell parts possible. Basically, however also several parts of a preparation at the same time with different ones specifically attached dyes are colored (multiple fluorescence). To differentiate between those emitted by the individual dyes  Fluorescence signals in turn become special dichroic beam splitters used.

In addition to excitation of the dye molecules with a high-energy photon (single-photon absorption), excitation with several photons of lower energy is also possible ( Fig. 1b). The sum of the energies of the individual photons corresponds approximately to that of the high-energy photon. This type of excitation of the dyes is referred to as multiphoton absorption (Lit .: Corle, Kino; "Confocal Scanning Optical Microscopy and Related Imaging Systems"; Academic Press 1996). Fig. 1b shows an excitation by means of the simultaneous absorption of 2 photons in the near infrared wavelength range. However, the dye emission is not influenced by this type of excitation, ie the emission spectrum experiences a negative Stokes shift during multi-photon absorption, ie it has a shorter wavelength than the excitation radiation. The excitation radiation is separated from the emission radiation in the same way as for single-photon absorption.

The state of the art will be explained below using the example of a confocal laser scanning microscope (LSM) ( Fig. 2).

An LSM is essentially divided into 4 modules: light source L, scan module S, detection unit DE and microscope M. These modules are in following described in more detail. It will also be on DE 197 02 753 A1, US 6167173 referenced.

For specific stimulation of the different dyes in one preparation are used in an LSM laser with different wavelengths. The The choice of the excitation wavelength depends on the absorption properties of the dyes to be examined. The excitation radiation is generated in the light source module L. Are used here various lasers A-D (Argon, Argon Krypton, TiSa laser). Still done in the light source module L the selection of the wavelengths and the setting the intensity of the required excitation wavelength, e.g. B. through use  an acousto-optical modulator. Then the laser radiation arrives via a fiber or a suitable mirror arrangement in the scan module S. The laser radiation generated in the light source L is with the help of the lens (2) diffraction limited via the scanner, the scan lens and the tube lens in the specimen ((sample 3) is focused. The focus is grid-shaped in x-y- Direction moved over the sample 3. The pixel dwell times when scanning over the sample 3 are usually in the range of less than a microsecond to to a few seconds.

With confocal detection (descanned detection) of the fluorescent light the light that comes from the focal plane (sample 3) and from the and is emitted below levels, via the scanner on one dichroic beam splitter (MDB). This separates the fluorescent light from the Excitation light. Then the fluorescent light is over dichroic Beam splitter DBS 1-3 and pinhole optics on one aperture (confocal aperture / Pinhole) (PH 1, 2, 3, 4), which is exactly in one to the focal plane of the Objective 2 conjugate plane is located. This will Fluorescent light components suppressed out of focus. By varying the Aperture size can adjust the optical resolution of the microscope become. Another dichroic block filter is located behind the aperture (EF1-4) which again suppresses the excitation radiation. After passing The block filter turns the fluorescent light into a spot detector (PMT1-4) measured.

When using a multiphoton absorption, the excitation occurs Dye fluorescence in a small volume in which the Excitation intensity is particularly high. This area is insignificant larger than the detected area when using a confocal Arrangement. The use of a confocal aperture can thus be omitted and the Detection via T-PMT, PMT 5 can be done directly after the detection direction Lens or on the side facing away from the lens (non descanned detection).

In a further arrangement for detection (not shown) one by one Multi-photon absorption of excited dye fluorescence continues a descanned detection, but this time the pupil of the lens from  the pinhole optics PH are imaged in the detection unit (non-confocal descanned detection).

From a three-dimensionally illuminated image is shown by the Detection arrangements in connection with the corresponding single photons or multi-photon absorption only the plane (optical section) reproduced, which coincides with the focal plane of the lens. By the recording of several optical sections in the x-y plane in different depths z of the sample can then be computer-aided three-dimensional image of the sample can be generated.

The LSM is therefore suitable for examining thick specimens. The The dye used with its excitation wavelengths specific absorption properties determined. On the emissions properties of the dye matched dichroic filters ensure that only the fluorescent light emitted by the respective dye from Point detector is measured.

Instead of a point scanner, so-called line scanners are also used according to the prior art (lit .: Corle, Kino; "Confocal Scanning Optical Microscopy and Related Imaging Systems"; Academic Press 1996). The basic structure corresponds essentially to that of an LSM according to Fig. 2. However, instead of a point focus, a line is mapped into the sample (3) and the sample to be examined is only scanned in one direction (x or y). The line focus is generated with the aid of at least one cylindrical lens (ZL) shown in broken lines in FIG. 2 in the collimated illumination beam path, in the focal length of which there is a pupil plane of the microscope arrangement. In such a construction, a slit diaphragm instead of the pinholes PH 1-4 serves as the confocal diaphragm (PH). This arrangement can also be used for undescanned detection when using a multiphoton absorption. Again, the confocal aperture (PH) can be omitted. A CCD camera (not descanned) or line (descanned) with 1024 or more pixels is used for detection instead of the point detector (PMT). By scanning a line instead of a point, the image acquisition rate can be increased significantly. This scanning method can therefore be used to observe fast-running processes in real time (real-time microscopy).

A disadvantage of this method is that the depth resolution by the Slit diaphragm is 1.4 times worse compared to point scanning laser scanning microscopes. This is because the confocal slit only fluorescent light outside the confocal Cut perpendicular to the scan line suppressed. In addition, deteriorates the lateral resolution.

In a further arrangement for real-time microscopy according to the prior art The entire field to be examined is examined using a technique flared light source illuminated. However, only special ones Dot pattern of the entire field to be scanned by a quickly rotating disc released. These procedures are mostly in the literature referred to as the Nipkow disk method (Lit .: Corle, Kino; "Confocal Scanning Optical Microscopy and Related Imaging Systems "; Academic Press 1996).

In another state-of-the-art method, the so-called structured illumination (see Fig. 3), the modulation depth of the optical image of an amplitude structure (e.g. grating) is used as a criterion for the depth of field. The image of the periodic structure is characterized by the frequency of the modulation and the phase position (image phase) of the modulation.

By a phase shift of the structure perpendicular to the optical axis different projection scenarios can be obtained.

In order to be able to calculate streak-free, depth-discriminated optical sections, at least 3 phase images PB at 0 °, 120 ° and 240 ° are generally required. These phase images (PB) are then calculated into a (confocal) optical sectional image in an image processor using the following formula:

where I (x, angle) is the intensity at the respective pixel in the corresponding one Phase image describes.  

The simplest case is to record the 3 or more phase images sequential. It is assumed that the sample is Measurement of the images not moving. The so calculated from the phase images Cross-sectional images or batches can then be created using 3-D Evaluation software can be displayed on a standard PC and monitor. The spatial resolution along the optical axis depends on the wavelength of light, the numerical aperture of the lens and the Modulation frequency from.

For a detailed description of the calculation algorithm, see T. Wilson et al .; "Method of obtaining optical sectioning by using structured light in a conventional microscope "; Optics Letters 22 (24) 1997.

Another disadvantage of previous methods for real-time microscopy is that that with a simultaneous examination of several dyes too several detection devices are to be provided. This increases the Data transmission requirements and the cost of such Device. That is why nowadays only microscopes become sequential Imaging of the different dye fluorescence used. In DE 198 29 981 A1 a method is described, the excitation wavelength and / or change intensity during the scan.

The object of the invention is to create a new method with Preparations are imaged in real time and with a high optical resolution can be.

This task is accomplished through arrangements and procedures according to the independent claims solved.

Preferred developments are the subject of the dependent claims.

The invention is for use in multi-fluorescence applications require, particularly suitable, since these simultaneously and advantageously also with optical resolution comparable to that of a point-scanning LSM, however, can be recorded in real time.  

The method can be used in imaging microscopy systems such as laser scanning microscopes for the three-dimensional examination of biological preparations with an optical, spatial resolution up to 200 nm and in analytical microscopy systems such as Fluorescence correlation spectrometers are used.

Furthermore, screening methods are based on fluorescence detection of dyes such. B. included in so-called chip readers differ from the processes of laser scanning microscopy distinguish significantly larger image field.

Description of the invention

The object of the method for increasing the spatial detection resolution, preferably in the case of a real-time imaging method, is a linear excitation and detection of the excitation radiation and / or excited fluorescence reflected, scattered and / or transmitted by a sample. For this purpose, a line focus of the excitation radiation is generated in the sample by means of suitable optics described in more detail below. Fig. 4 shows a possible measurement flow diagram. A scan field with the line focus is shown on the left. The line focus can be shifted along the arrow with suitable tools (scanner mirror in one direction). The axial and lateral optical resolution is increased by structuring the line focus. The structuring takes place by overlaying the scan line with a periodic structure, for example generated by a sine grating in the beam path. In the case of coherent illumination, the structure can also be generated by an interference of 2 coherent partial beams in the sample (lit .: Wilson WO 98/45745). At least two images with different phase positions of the periodic structure are recorded. In principle, the optical cut can also be made by taking images with different modulation frequencies of the structure.

The sample illumination can be spatially and temporally coherent or incoherent lighting. For example, it can also be a  spatially coherent laser light source by using a Lens or by lining up several individual foci in the Sample can be used for incoherent lighting.

There are advantageously at least 2 phase images PB with image phases of z. B. 0 ° and 180 ° when using an excitation wavelength for the calculation an optical sectional image in the image processor is required.

With n excitation wavelengths n + 1 phase images are necessary.

For an excitation wavelength, the signal emitted by the sample along a scan line has the following form:

I _Sig (x) = C (x) .cos (kx) + B (x)

where C (x) carries the actual object information (e.g. the concentration and cross-section of the dye or the local reflection coefficient) at position x within the confocal optical section and along the line focus for the excitation wavelength and represents the size sought. B (x) carries the object information from outside the confocal area. B (x) is not modulated because the structure outside the confocal area is not sharply imaged. k is the inverse modulation frequency of the structured excitation. The above equation has 2 unknowns C (x) and B (x). To determine C (x) it is necessary to take two phase images. Let I _i (x), i = 0, 1,. , , designated n and characterized by a relative phase shift of the modulation of i.Δϕ (image phases), resulting in the following system of equations.

I ₀ (x) = C (x) .cos (kx) + B (x)

I ₁ (x) = C (x) .cos (kx + Δϕ) + B (x)

The object information we are looking for is obtained by solving the system of equations according to C as follows:

Analogously, the determination of C (x) can also be done by taking 2 pictures with different modulation frequencies k ₀ and k ₁ . The frequency images are again with I _i (x), i = 0, 1,. , , n denotes, resulting in the following system of equations.

I ₀ (x) = C (x) .cos (k ₀ .x) + B (x)

I ₁ (x) = C (x) .cos (k ₁ .x) + B (x)

The object information we are looking for is obtained by solving the system of equations according to C as follows:

C (x) can also be done by taking at least two pictures with different Phase and frequency of the modulation can be obtained.

From this line-related object information, an optical section in composed of at least one sample level and, as in the prior art Technology for at least one image of at least one image stack to be processed further.

If only one excitation wavelength is used and at least 3 Phase images (e.g. with relative phase positions of 0 °, 120 ° and 240 °) can also be recorded, that explained in the prior art Algorithm for obtaining the object information (Lit .: T. Wilson et al .; "Method of obtaining optical sectioning by using structured light in a conventional microscope "; Optics Letters 22 (24) 1997) in all here described arrangements apply.

With simultaneous excitation with several wavelengths, the subsequent separation of the signals of the sample by a special one Phase coding or frequency coding of the modulation structure for the different excitation wavelengths. In general, n results in simultaneous Excitation wavelengths the number of required phase images to n + 1. Different excitation wavelengths are defined by one wavelength-specific position shift of the modulation (coding phase) and / or a respective characteristic modulation frequency (Coding frequency) separated from each other. The light emitted by the sample (e.g. fluorescence), which is caused by an interaction of the excitation light of the respective wavelength with the sample then has the specific coding phase and / or coding frequency depending on the Excitation wavelength and can thus be according to the following Algorithm can be separated.  

So is z. B. a sample, as shown in Fig. 5, colored with different dyes (1, 2, 3) in the scan field, which require different wavelengths (L ₁ , L ₂ , L ₃ ) to excite fluorescence, the coding frequency is transmitted (f ₁ , f ₂ , f ₃ ) and / or the coding phases (ϕ ₁ , ϕ ₂ , ϕ ₃ ) of the excitation scan line for the detected fluorescence in the respective region (1,.. n), see Fig. 6.

In the preferred case, all excitation wavelengths have the same coding frequency, ie a modulation frequency but different coding phases. The following discussion is limited to this case with phase coding, with coding using the modulation frequency to be treated analogously and the subject of the invention. For two excitation wavelengths, the signal emitted by the sample along a scan line has the following shape:

I _Sig (x) = C ₁ (x) .cos (kx + ϕ ₁ ) + B ₁ (x) + C ₂ (x) .cos (kx + ϕ ₂ ) + B ₂ (x)

where C ₁ (x) and C ₂ (x) represent the object information (e.g. concentration and cross section of the dye) at position x within the confocal optical section and along the line focus for the two excitation wavelengths. B ₁ (x) or B ₂ (x) carries the object information from outside the confocal area. B ₁ (x) and B ₂ (x) are not modulated because the structure outside the confocal area is not sharply imaged. ϕ ₁ and ϕ ₂ are the coding phases assigned to the respective excitation wavelengths and k is the inverse modulation frequency (which is the same for both excitation wavelengths in this example) of the structured excitation. The above equation has 4 unknowns C ₁ (x), B ₁ (x), C ₂ (x) and B ₂ (x).

To determine C ₁ (x) and C ₂ (x), three phase images must be taken. Let I _i (x), i = 0, 1,. , , designated n and characterized by a relative phase shift of the modulation of i.Δϕ (image phases), resulting in the following system of equations:

I ₀ (x) = C ₁ (x) .cos (kx + ϕ ₁ ) + B ₁ (x) + C ₂ (x) .cos (kx + ϕ ₂ ) + B ₂ (x)

I ₁ (x) = C ₁ (x) .cos (kx + ϕ ₁ + Δϕ) + B ₁ (x) + C ₂ (x) .cos (kx + ϕ ₂ + Δϕ) + B ₂ (x)

I ₂ (x) = C ₁ (x) .cos (kx + ϕ ₁ + 2.Δϕ) + B ₁ (x) + C ₂ (x) .cos (kx + ϕ ₂ + 2.Δϕ) + B ₂ (x)

The object information we are looking for is obtained by solving the system of equations according to C ₁ and C ₂ as follows:

C ₁ (x) = [-c ₁₂ (x). (I ₂ (x) - I ₀ (x)) + c ₂₂ (x). (I ₁ (x) - I ₀ (x))] / det (x)

C ₂ (x) = [c ₁₁ (x). (I ₂ (x) - I ₀ (x)) - c ₂₁ (x). (I ₁ (x) - I ₀ (x))] / det ( x)

with c _ij (x) = cos (kx + ϕ _j + i.Δϕ) - cos (kx + ϕ _j )
and det (x) = c ₁₁ (x) .c ₂₂ (x) - c ₁₂ (x) .c ₂₁ (x). Obtaining the object information therefore requires knowledge of the coding phases (ϕ _i ) and the inverse modulation frequency (k) for the individual excitation wavelengths. With a two-color excitation, three phase images are required to solve the system of equations and thus the separation of the object information, which has been made visible by the respective excitation wavelength, the image phase shift (Δϕ) between the individual images being 120 ° in the simplest case.

The object information from outside the confocal area can only be obtained for the sum of the contributions of all exciting wavelengths. In the present example this is the background

B ₁ (x) + B ₂ (x) = I ₀ (x) - C ₁ (x) .cos (kx + ϕ ₁ ) - C ₂ (x) .cos (kx + ϕ ₂ )

which can be calculated with knowledge of C ₁ and C ₂ .

The extension to 3 excitation wavelengths is equivalent to the solution of the corresponding system of equations, whereby C ₃ (x) or B ₃ (x) should represent the object information stimulated by the third wavelength from inside or outside the confocal cut. The object information for the individual wavelengths is as follows:

C ₁ = [(c ₁₂ .c ₂₃ - c ₁₃ .c ₂₂ ). (I ₃ - I ₀ ) + (c ₁₃ .c ₃₂ - c ₁₂ .c ₃₃ ). (I ₂ - I ₀ ) + (c ₂₂ .c ₃₃ - c ₂₃ .c ₃₂ ). (I ₁ - I ₀ )] / det

C ₂ = [(c ₁₃ .c ₂₁ - c ₁₁ .c ₂₃ ). (I ₃ - I ₀ ) + (c ₁₁ .c ₃₃ - c ₁₃ .c ₃₁ ). (I ₂ - I ₀ ) + (c ₂₃ .c ₃₁ - c ₂₁ .c ₃₃ ). (I ₁ - I ₀ )] / det

C ₃ = [(c ₁₁ .c ₂₂ - c ₁₂ .c ₂₁ ). (I ₃ - I ₀ ) + (c ₁₂ .c ₃₁ - c ₁₁ .c ₃₂ ). (I ₂ - I ₀ ) + (c ₂₁ .c ₃₂ - c ₂₂ .c ₃₁ ). (I ₁ - I ₀ )] / det

with ϕ ₃ (x) as the coding phase of the modulation at the third excitation wavelength and

det (x) = c _11. (c ₂₂ .c ₃₃ - c ₂₃ .c ₃₂ ) + c _12. (c ₂₃ .c ₃₁ - c ₂₁ .c ₃₃ ) + c _13. (c ₂₁ .c ₃₂ - c ₂₂ .c ₃₁ ).

With 3-color excitation, 4 phase images must be recorded to separate the object information, stimulated by the individual wavelengths. The image phase step size can be 90 degrees, for example. Since the quantities c _ij are constant for an arrangement with predetermined coding phases and coding frequencies, obtaining the object information by signal processing is a simple operation. This operation can be specified by transforming the normalized image signals ΔI _i (x) = I _i (x) - I ₀ (x) by means of factors f _k (x) stored as constants, which result from the c _ij (x). For 3 wavelengths z. B. from the above equations:

C ₁ = f ₁ .ΔI ₃ + f ₂ .ΔI ₂ + f ₃ .ΔI ₁

C ₂ = f ₄ .ΔI ₃ + f ₅ .ΔI ₂ + f ₆ .ΔI ₁ .

C ₃ = f ₇ .ΔI ₃ + f ₈ .ΔI ₂ + f ₉ .ΔI ₁

In general, for simultaneous excitation with n wavelengths, it is necessary to record n + 1 images I ₀ to I _n , each with its own phase and / or frequency of the excitation modulation. In the case of phase images (image phase: ϕ _i ) with phase coding of the excitation wavelength (ϕ _j ), the following images result which contain the image information of the pseudo-confocal cut (C _j ) and of the background (B _j ) excited with the respective jth wavelength.

The resulting system of equations of form:

with c _ij (x) = cos (kx + ϕ _j + ϕ _i ) - cos (kx + ϕ _j ), known mathematical methods (e.g. according to Cramer's rule) can be used to find the image information sought from within the pseudo-confocal cut, separate according to the individual excitation wavelengths.

Furthermore, the image information excited by all wavelengths can be read from outside the pseudo-confocal section

determine.

Analogous relationships apply if, instead of or in addition to the phase, the frequency is used to encode the different wavelengths and / or the images. The coefficients c _ij can then be modified appropriately.

The coefficients c _ij can be determined using the measuring method described below, regardless of whether the phase and / or the modulation frequency are used for image or wavelength coding. For a reference wavelength (for example 488 nm), the coding frequency and the coding phase are determined in the sense of a calibration (for example by means of a test object such as a glass plate or a mirror). For this purpose, the periodic structure is mapped onto the test object, measured with the detector, and the coding frequency and the coding phase for a fixed image phase are measured at the location of the sample and thus determined. The coding phases or coding frequencies of the other wavelengths can now in turn be measured relative to this reference for a fixed picture phase. In one possible arrangement, phase coding is implemented by dispersion (parallel offset) in a plane-parallel plate P, which at the same time represents the support of an amplitude grating (structure S) arranged in front of it in the direction of illumination, see Fig. 7A. The passage of polychromatic excitation light through the plane-parallel plate P (inclination: γ, thickness: d), which is slightly inclined around axis A, results in a slight, wavelength-dependent parallel offset in the plane perpendicular to the axis of rotation and the optical axis:

Δx (λ) = d. (Sin γ - cos γ.tan β (λ)) with β (λ) = sin ^-1 ((sin γ) / n (λ)).

If the plate is in an intermediate image plane (see Fig. 9A) of the microscope arrangement, this offset manifests itself as a phase shift between the amplitude modulations of the individual excitation line foci. Fig. 8 schematically shows a line focus in a scan field, the phase positions of the amplitude modulation for, for example, 3 different excitation wavelengths being shown in the diagram on the right. The x-axis of the diagram corresponds to the coordinate along the line focus. In the right figure of Fig. 8 only a section of the scan line is shown to clarify the coding phases.

A phase shift of 5 degrees is sufficient for safe separation the signals excited by the corresponding wavelength. Table 1 gives the coding phases for a concrete example.

Table 1

Phase shift for important laser lines through dispersion in a 10 mm thick BK7 Plate with an amplitude grating of 50 lines / mm. The plate is 5 degrees against the normal inclined so that there is a parallel offset of approx. 300 µm for the reference wavelength of 488 nm compared to the optical axis.

An arrangement for wavelength-dependent phase shift (Phase encoding) uses an optical grating, which is in a Pupil level of the microscope arrangement - for example in the scanner is used.

In the following, advantageous arrangements are exemplified for fluorescence and DIC imaging are explained without being transferable to others optical contrast method is limited.

Arrangement 1 Structured lighting with single scan line

A first arrangement is shown in Fig. 9. The pupil beam path of the microscope arrangement is shown in dashed lines. Solid lines represent the object beam path. In FIG. 9A (xy plane), light from the light source (LQ) with, for example, a cylindrical lens (ZL), in the focal length of which there is a pupil plane of the microscope arrangement, becomes a line focus in an intermediate image plane (ZB) shaped. In an intermediate image plane in front of or behind the cylindrical lens (shown in front of the cylindrical lens) there is an element (ST) which causes amplitude modulation along the scan line. For example, ST is a transmission grating. ST is, for example, a transmission grating. As shown by way of example in FIGS. 7B / 7D, the change in the periodicity runs perpendicular to the direction (Y) of the grid lines shown schematically as black lines in the X direction. A shift in this X direction, for example with the positioning unit PE as shown in Fig. 9, enables a change in the phase position. A shift of the structure according to Fig. 7D in the Y direction (shift not shown) results in a variation in the frequency of the amplitude modulation to adjust the optical section thickness (see text below) or to change the modulation frequency when taking the individual images to calculate the optical sectional image. Tilting about a pivot point that lies in the optical axis and an axis of rotation in the Y direction also produces this displacement. A rotation of the grating about an axis of rotation in the Z direction, which for example coincides with the optical axis, also produces (see Fig. 16) a phase shift that turns out differently when several spatially separated lines are mapped onto the sample (line profile in the X direction).

In the following, the structured scan line is scanned by the main beam splitter (MDB) and a scanner (y), which moves the scan line perpendicular to the line (y coordinate) and is in turn in a pupil of the microscope arrangement, through the scan optics (SO), the tube lens (TL) and the lens (O) imaged in the sample (PR). The relay optics (RL) represent the focal point of the cylindrical lens (ZL) on the scanner (y). The relay optics can also be omitted in special arrangements according to the prior art. For example, it can be omitted if the distance between the two scanners x and y is shortened or if the scanners x and y are replaced by a gimbal-mounted individual scanner. In principle, the cylindrical lens can also be replaced by a cylindrical mirror whose focal point lies on the scanner (X). The cylinder mirror is arranged at 45 ° in the xz plane shown in Fig. 9C. At this level, the mirror also has its focusing effect. Furthermore, the beam path to the light source is angled through 90 ° through the mirror. This is shown schematically in Fig. 9c.

The emission light collected by the lens (O) is with the help For example, the main color splitter (MDB) is split off from the excitation light. Subsequently, the light of the sample is measured using an imaging optics (PO) with confocal detection through a slit diaphragm (position slit) Longitudinal direction in the Z direction on the drawing) focused, whereby Fluorescence that is out of focus is suppressed. At a the aperture can be omitted for unscanned detection. Behind the slit diaphragm there is a line or area detector (DE) (position of the line in Z- Direction) of the spatially resolved (along the line focus) the fluorescence detected. In addition, an emission filter (not shown) for Suppression of the excitation radiation in the detection beam path after the Main color dividers (MDB) can be arranged. The line focus is with the Galvo scanner (y) scanned in one spatial direction. If the x-scanner is not used to adjust the image phase, it remains in its Zero position.

Fig. 9B shows the same arrangement but this time mirrored by 90 degrees (YZ plane). Instead of a line on the sample, a focus point on the sample can be seen in this spatial direction due to the cylindrical lens (ZL) not acting in this direction.

In principle, the use of the slit diaphragm can also be dispensed with. This is particularly useful if the imaging of the fluorescence is not to be scanned via the scanner, but is to be done directly on a CCD camera or a gated camera for spatially resolved measurement of the fluorescence lifetime (LaVision, Picostar). The CCD camera looks at an intermediate image plane ZB, for example. B. in the intermediate image plane between TL and SO of the microscope arrangement. On a microscope according to the state of the art, it is connected to a TV port ( Fig. 19 TV port position). In addition, a CCD camera can be used in the transmission beam path of the microscope arrangement (e.g. Fig. 1 instead of T-PMT). As a result, the excitation radiation transmitted by the sample can be detected with confocal resolution. Confocal DIC mapping is also possible. For this purpose, the DIC prism is attached in the objective pupil between TL and objective O according to the prior art. The image obtained in this way with DIC can also be displayed in an image superimposed on the images generated by signals reflected, scattered and / or emitted by the sample. The confocal cut is calculated, for example, by taking at least two phase images (see Fig. 4, or in the text above). The lighting intensity of the lines in the at least 2 phase images varies periodically in the x direction - for example sinusoidally - due to the structure ST. The relative image phase when using 3 structured lines is shifted by 120 ° phases. The 3 phase images are generated sequentially, ie 3 images are scanned in succession using the y-galvo scanner (y). The structure (ST) can be a transmission grating, for example. The image phase can be shifted, for example, with a positioning unit (PE) to which the transmission grating is coupled, so that each location of the sample to be examined can be viewed with varying brightness. The setting of the image phase can also be carried out at a wavelength with the help of a rotatably mounted plate (PL) as in Fig. 7A, which is upstream / imprinted on the structure. For this purpose, the plate is to be arranged in an intermediate image plane of the microscope arrangement, as shown in Fig. 9.

The plane-parallel plate used here is located in an intermediate image plane, the structure ST (shown in FIG. 7B) is preferably applied directly to the plate.

In a further arrangement, the adjustment of the image phase can also be done with the help another galvo scanner (X). The scanner (X) adjusts here the image phase by moving the scan line in the x direction. This has the Advantage that by switching on (swiveling in or pushing in) the  Cylinder lens (ZL) and the transmission grating coupled with a detection-side beam deflection for switching from one Point detector on a line detector (DE) between one point-scanning LSM and a line-scanning LSM with structured Lighting can be switched. If the scanner X is not Setting the image phase is used, it remains in its Zero position (is therefore de-energized).

Arrangement 2 Structured lighting with single scanline and Coding of the modulation phase or modulation frequency

A second arrangement uses different coding phases depending on the excitation wavelength used when structuring the scan line. As a result, the signals of the fluorescence excited by the individual wavelengths can be digitally separated. Fig. 10A shows a possible schematic structure in the XZ plane. Light from the light source (LQ) of the different wavelengths undergoes amplitude modulation with the aid of a structure (ST). For this purpose, the structure is located in an intermediate image plane of the microscope arrangement. The structure is displaceably arranged via a positioning unit (PE), so that the structure can be moved along the scan line and different image phases can be set. The structured light is then shaped in a cylinder optics (ZL) into a line in the pupil plane of the microscope arrangement. For this purpose, the cylindrical lens (ZL) is arranged at a distance of its focal length from the pupil (scanner X). The setting of the image phase can also be carried out with the scanner (X), which is located in a pupil plane of the microscope arrangement.

Fig. 10B shows a simplified arrangement with only one scanner (Y) in the XZ plane. The setting of the image phase is done by the positioning unit PE.

After an amplitude modulation has been impressed on the scan line, the radiation passes through an element in which the structure is shifted depending on the wavelength (ST) and thus the coding phase is varied depending on the wavelength. A possible arrangement for phase coding has already been described with reference to Fig. 7A. The plane-parallel plate used here is located in an intermediate image plane, the periodic structure ST (shown, inter alia, in FIG. 7B) is preferably applied directly to the plate.

Following the phase or frequency coding of the The excitation radiation passes through the amplitude modulation Main color splitter (MDB), the Y scanner, the scanning optics (SO), the tube lens (TL) and the lens (O) in the sample (PR). The Y scanner rasterizes the Scan line for recording a phase image in the y coordinate. In Depends on the dyes used Fluorescence excitation with the corresponding wavelengths. The Modulation frequency and modulation phase of the scan line depending of the excitation wavelength is thus transferred to the Fluorescent signal. In the case of a scanned detection, the fluorescence collected by the lens and get through the tube lens, scanning optics and the Y scanner to the main color splitter (MDB). This separates the fluorescence from the excitation radiation. The fluorescence is then switched off by a Slit diaphragm mapped onto a line detector (DE). For suppression The excitation radiation can additionally block filters in front of the line detector be switched. The slit diaphragm can in particular with a Multi-photon excitation is eliminated. The separation of the individual signal components, generated by the different wavelengths, and the The optical sections are calculated using the method described above Algorithm, for example, using the coding phases for the same Modulation frequency, the coding phases of the individual wavelengths with test objects such as plane mirrors have been determined beforehand. It can thus regions that were colored with different dyes are separated be made visible.

Another arrangement uses a dispersive element (DG), e.g. B. a transmission grating, which is located in a pupil plane of the microscope arrangement. The optics diagram is shown in Fig. 11 in the XZ plane. The transmission grating shifts the individual scan lines according to their wavelength within the drawing plane (only one WL is shown). As a result, a different phase position of the modulation structure ST (coding phase) is impressed on the individual scan lines according to their wavelength. The image phase is shifted with the aid of the positioning unit (PE) and the modulation structure S for each wavelength with an identical step size. This ensures that the image phase shift for each excitation wavelength is 120 °, for example when 3 phase images are taken.

In the following, advantageous arrangements for generating the Frequency coding or the adjustment of the optical resolution closer received.

By changing the modulation frequency for each excitation wavelength the effective pinhole size, i. H. the optical section thickness for the individual wavelengths can be adjusted.

Fig. 7C schematically shows a device for wavelength-dependent adjustment of the modulation frequencies, which is advantageously installed in arrangements 1 and 2 (as for example in Fig. 9) in an intermediate image plane of the microscope device instead of the structure ST. A polychromatic light source with a dispersive element (DG1) is spatially split into its spectral components. The spectral components are then made axially parallel with a first lens (lens 1, focal length f), ie DG1 is at the focal point of the lens. The structure (ST) with which the amplitude modulation takes place is located in the imaging-side focal plane of the lens. A possible formation of the structure with a linear dependence of the frequency in the y direction is shown schematically in FIG. 7D.

The division according to FIG. 7C) into lines L1, L2, L3 produces on this periodic structure due to the different position of the lines in y- Direction of different modulation frequencies.

So there is a spatial splitting linearly dependent on the wavelength the polychromatic light source, e.g. B. with a transmission grid, so With a suitable grid design, the modulation frequencies also change of the individual spectral components as a function of the wavelength. This can, for. B. ensure that the same optical Cutting thickness is realized at different wavelengths. In connection the individual spectral components are measured using a second lens (lens 2, Focal length f, which need not be identical to the focal length of lens 1) and  another dispersive element (DG2, e.g. transmission grating that does not must be identical to DG1) again spatially superimposed. By customization the focal lengths of lenses 1 and 2 can control and expand the beam so that the illumination of the microscope objective can be optimized.

Overall, the following advantageous variants of the invention result from such a structure:

• a) By shifting in the x-direction of the structure ST, a different phase change (based on a previous calibration measurement) can be generated for the individual lines, with a complete scanning of the sample using the respective line taking place in the different phase positions and wavelength-dependent sectional images being calculated ,
  The slice thickness can be changed by a defined shift of the frequency by shifting in the y direction.
• b) The (modulation frequency) is changed in a defined manner by (at least one) shift in the y direction, and thus sectional images can be calculated for the individual wavelengths by repeated scanning at different modulation frequencies.
  Via the different modulation frequencies of the structure, frequency coding / phase coding for different wavelengths can also be generated when all wavelengths are mapped on a common line (see arrangement 4).

Instead of the structure shown in Fig. 7D with a continuous change in the modulation frequency in the y direction, a structure (see also Fig. 16) that is composed of several substructures of different modulation frequencies can be used. However, this has the disadvantage that the structure is only adjusted for fixed excitation wavelengths. In the simplest case, however, different gratings ST, each with a different modulation frequency, can be pivoted into the arrangements 1 and 2.

In a further arrangement, not shown, the radiation passes after Structuring an element in which the structure is wavelength dependent  enlarged and thus the modulation frequency varies depending on the wavelength, d. H. frequency coding is carried out. In a first order the scan line is separated into its individual by means of dichroic beam splitters Wavelength portions broken down and then in the partial beam paths wavelength-dependent magnification with one telescope each Imprinted zoom optics to adjust the magnification. With the help of others The individual scan lines are again superimposed on dichroites. As alternative can in a further arrangement also special diffractive elements generate a wavelength-dependent enlargement of the scan line, be used.

Depending on the wavelength used, the scan line has a specific coding frequency of the amplitude modulation at the output. The image phase is shifted with the aid of the positioning unit (PE) and the displaceable structure ST for each wavelength with an identical step size. This ensures that the image phase shift for each excitation wavelength is 120 °, for example when 3 phase images are taken. Instead of taking phase images, the optical section can also be calculated from two images in which the modulation frequency per excitation line is different in the two images. The modulation frequency can be changed for all or one wavelength by taking the two images at different positions in the y-direction of the transmission grating according to FIG. 7D.

It can by the arrangements described above z. B. ensure that the axial optical resolution of the microscope is the same for different wavelengths. This enables so-called colocalization measurements with a line scanner. In addition, e.g. For example, in the case of very weak fluorescence signals, the effective pinhole can be enlarged by moving the structure in the Y direction, see Fig. 7D, here downwards. Although this reduces the optical resolution, the signal to noise ratio can be improved.

In addition, the above-described zoom optics, which are not shown, can do this serve the chromatic longitudinal error of the imaging optics compensate and thus ensure that the optical cut in the the same object level takes place. For this purpose, one lens each  individual zoom optics slightly depending on the used Microscope lens misaligned so that the beam for the individual Wavelengths at the output of the zoom optics divergent or convergent on the Lens pupil hits and thereby the focus depending on the Excitation wavelength shifted in the axial direction, d. H. for all Excitation wavelengths can be covered.

In addition to versions 1 and 2, another dispersive element (PR) can be used for the additional spectral splitting of the fluorescence signals perpendicular to the illumination line (multispectral line scanner). Fig. 12 shows the detection scheme of a CCD area of the multispectral line scanner in the XZ plane. The structured emission signals for the respective wavelengths are imaged in the x direction of the detector (DE).

The setting can be used as an alternative to shifting the phase position using PE the image phase is also done here by the X scanner.

For each scan position of the scan line, an image is taken as shown for different wavelengths of detection. The freely programmable control of selected detector elements (rows) and the combination of detector elements in one column of the detector matrix allows a flexible selection of spectral ranges of the fluorescence emission. For this purpose, several lines of the detector, in which different fluorescence signals are mapped according to their wavelength, can be connected electronically. The multispectral detection of the fluorescence emission can in turn be combined with the structured lighting. For this purpose, the linear excitation is additionally z. B. structured by means of a transmission grating (ST). Here, a complete scan of the scan line takes place, each with the detection of the wavelength distribution corresponding to the illustration in FIG. 12, and further scans with a change in the image phase of the grating structure. Since, for example, a plane plate PL used was tilted in a defined manner to determine a wavelength-dependent offset, the phase position can be changed, for example, by shifting with PE at a constant tilt angle. The depth contrast can be optimized again by sequential detection of images of structured objects with different image phases of the excitation and subsequent calculation. Fig. 12 shows the combination of a multispectral line scanner (spectral splitting takes place in the y-plane into the drawing plane) with structured illumination via the structure ST, the phase coding in turn with a tilted and parallel to the optical axis plane-parallel plate ( not shown here) or with a dispersive element (see text above). The image phase is varied sequentially. The different spectral components are detected in parallel using a CCD array.

The slit diaphragm (SB) used here as the entry slit of the spectral splitting serves, can be omitted in the case of a scanned detection in the line scanner. Thereby the spectral resolution decreases, but the technical resolution Effort significantly reduced, as otherwise slidable single slot covers must be used.

Fig. 13 shows schematically the structure of the electronics for the detection of the signals with a line detector. For this purpose, signals from the detector K1-N are amplified with the aid of a respective current-voltage converter (A) and then integrated during the pixel dwell time (I). After further amplification for level adjustment to an analog / digital converter (ADC), these are converted into digital signals in the ADC and transmitted to a computer (PC). The detection with a CCD matrix is known from the literature.

Arrangement 3 Structured lighting with quasi-simultaneous recording the live images

The striped images can also be detected quasi-simultaneously by imaging several lines (e.g. L1, L2, L3) in the sample instead of one line, the intensity structure (image phase), for example analogously to FIG . B. 120 ° when using three lines, for example ( Fig. 14). Thus, when the sample is scanned once, the entire information for evaluating the data is generated. Fig. 15 schematically shows the optical structure in the YZ plane. The focal points along the y-axis of the line focus are drawn in each case. The excitation light can be split using a beam splitter arrangement (T). Two embodiments of the beam splitter arrangement (T) are shown in Fig. 16A / B, here upstream of the cylindrical lens. The first arrangement in cooperation with the cylindrical lens produces several discrete line foci with two mirrors arranged in parallel, the first mirror being a full mirror and the second mirror being a partially transparent mirror. The transmission of the partially transparent mirror is matched to each partial beam (i) and results from 100% / (ni), where n is the total number of individual foci. The portions (i) then arrive separately in a special lattice structure (ST), which is shown schematically in Fig. 16A. With this structure, the partial gratings are modulated out of phase in a defined manner. Instead of the structure composed of partial gratings, a transmission grating according to Fig. 7B, as shown in Fig. 16A, can also be used in a twisted manner, with which the phase shift of the structure for the individual scan lines can be set specifically for the same modulation frequency. The device is calibrated, ie the modulation frequency and the phase positions of the scan lines are determined using the method described above. The structure is located in an intermediate image plane of the microscope arrangement. The divider arrangement can also be used, for example, in a pupil plane of the microscope arrangement. Here, both mirrors are tilted against each other, so that again several line foci arise.

The second arrangement in Fig. 16 ( Fig. 16B) is a special arrangement for generating a double line focus. The arrangement in turn uses two mirrors (SP1 and SP2) arranged in parallel, one mirror (SP1) carrying the structure ST itself. The structure is reflective at the points of low transmission. The structure is arranged in the intermediate image of the microscope arrangement. At the output of the divider there are 2 lines, the amplitude modulation of which is exactly opposite in phase. Line 1 thus carries exactly one amplitude modulation shifted by 180 ° compared to line 2. The structure is applied to glass plate 1. If several wavelengths are used, the wavelength-dependent parallel offset in the y direction through the plane-parallel glass plate is compensated for by a second glass plate of the same thickness arranged at an angle of 90 °. In addition, the entire divider unit (T) can be easily tilted about the y-axis to generate the phase encoding described above. A shift of the structure (ST) can be omitted in both arrangements. The scanning movement of the Y scanner takes place beyond the actual image field to be examined, so that each point of the sample to be examined is illuminated once with a line of different phase position of the structure. This arrangement is particularly advantageous for the examination of rapidly changing samples, since movements or rapid changes in the sample no longer have a disruptive effect on the measurement and the maximum image recording speed of the line scanner is not reduced by the sequential data recording. The detection of the lines excited in parallel takes place here with a matrix detector, in the case of, for example, 3 lines with a 3-line detector. Alternatively, a line detector can also be used, on which all lines separated on the sample are detected simultaneously. The separation of the information from each line can be done by the algorithm described above based on the different modulation (in phase and 1 or frequency) of each line.

Arrangement 4 Structured lighting with quasi-simultaneous multispectral excitation

In a fourth arrangement, several discrete excitation wavelengths are imaged spatially separated into different scan lines (e.g. with 3 laser lines: L1, L2, L3 in the x direction) in the sample. For this purpose, a parallel dispersive element (PR) (e.g. transmission grating, grating lines in the x-direction) generates several parallel excitation lines of different wavelengths (see Fig. 17), which are transferred using the y-galvo scanner (y) and the following optical image the sample is moved so that every point in the interesting scan field is scanned at least once by each line. The optical structure is shown schematically in Fig. 18 in the YZ plane. The focal points of the focus lines are shown.

The fluorescence signals are split off from the excitation again with the main color divider (MDB). The fluorescence signals arrive  generated by the different excitation lines different positions L1-L3 in the z-direction of a matrix detector upstream slit diaphragms in the x direction, with the axis perpendicular to Illumination line of the corresponding wavelength of fluorescence equivalent. The axis along the illumination line on the matrix detector corresponds to the local coordinate.

In addition, phase or frequency coding can also be carried out in accordance with arrangements 1 and 2. For example, for phase coding, the structure (ST) is tilted about the Y axis. Frequency coding can e.g. B. with the special structure shown in Fig. 7D. When using phase or frequency coding, the spectral components can again be detected simultaneously with a detector matrix that detects all scan lines simultaneously. In the simplest case, a CCD line with rectangular pixels (or interconnected lines) is used for this, the longer sides of the pixels advantageously being oriented in the direction z (see FIG. 18), so that, for example, the scan lines L1 to L3 together with the detector line can be detected. The signals that were excited by the different scan line foci are then separated again using the algorithm described above. The advantage of these arrangements is that the individual scan lines no longer have to be detected separately on a detector array, but can instead be detected with a one-dimensional detector.

In addition, a further dispersive element can serve for the additional spectral splitting of the fluorescence signals perpendicular to the illumination line. Again, the detection scheme of the multispectral line scanner shown in Fig. 12 can be used. A spatial coordinate is imaged in one direction of the detector. The freely programmable control of selected detector elements (rows) and the combination of detector elements in one column of the detector matrix allows a flexible selection of spectral ranges of the fluorescence emission. For this purpose, several lines of the detector, in which different fluorescence signals are mapped according to their wavelength, can be electronically connected together. The multispectral detection of the fluorescence emission can in turn be combined with the structured lighting. For this purpose, the linear excitation is additionally z. B. structured by means of a transmission grid. The depth contrast can be optimized again by sequential detection of images of structured objects with different image phases and subsequent calculation. Fig. 18 shows the combination of a multispectral line scanner with structured lighting. The image phase is varied sequentially with different structures. The different spectral components are detected in parallel, for example, using a CCD array. According to the state of the art, a plurality of diaphragms arranged and conjugated to the individual scan lines and freely movable diaphragms would have to be used as confocal slit diaphragms, but these can be dispensed with by structuring the scan lines. This significantly simplifies the technical effort.

Arrangement 5 Structured lighting with punctiform Sample interaction

In a fifth arrangement, instead of a linear excitation of the sample, there is a point excitation of the sample. For this purpose, the laser scanning microscope according to Fig. 1 is operated according to the prior art. However, a structure (ST) according to Fig. 19 is used in an intermediate image plane, which is arranged in the sample direction after the scanners x and y. The structure is successively imaged in the sample by scanning in the x or y direction. The phase images are recorded by moving the structure with PE. In addition, quasi-simultaneous lighting according to arrangement 3 and 4 can also take place. Phase coding or frequency coding with the arrangements described above can be transmitted without restriction. The advantage of this arrangement compared to a conventional point scanner is that the signal of the sample can be measured directly with a CCD camera without descanning. In this case, the confocal aperture is also dispensed with. For example, a gated camera (Picostar, La Vision) can also be used as the CCD camera. In this way, for example, time-resolved fluorescence recordings of confocal optical sections can be achieved. According to the state of the art, this is only possible through the use of nonlinear sample interactions.

However, it can also be a descanned point-shaped detection or partial Descanned detection (Lit .: Stimson et al., Rev. Of Sc. Instr., (70), p3351, 1999) according to the state of the art.

By phase coding or frequency coding with the described detection variants continue to stimulate the signals of the sample through different wavelengths can be recorded simultaneously. It is only one detector required. Due to the arrangement, the structure of a laser scanning microscope can be significantly simplified without that flexibility is restricted. By turning on the structure can be between a conventional laser scanning microscope and a Laser scanning microscope with structured lighting back and forth be switched.

A combination of methods 1-5 is also part of the invention. Farther is also a combination of the methods described above so-called parallel scanning methods according to the prior art possible without restriction. Mapping takes place with these methods of several points or lines of the same intensity simultaneously as a matrix in the Probe (Lit .: Buist et al., J. or Microscopy (192), p217, 1998 / Nielsen et al., J. o. Microscopy (201), p852, 2000). This allows the image capture rates be increased again. Furthermore, the sample can be used with a Table scanner or a scanner according to the state of the art (e.g. Nipkow- Disc).

The advantageous arrangements described can also be used advantageously for examining macroscopic samples. Macroscopic samples are used, for example, when screening active substances on a chip. The edge length of the scan fields is some 10 mm. These scan fields can e.g. B. by increasing the scanning angle of the galvo scanner by arranging the sample in an intermediate image of the microscope arrangement, for example in FIG. 2, or by a special lens arrangement (macro objective), which images the intermediate image enlarged on the sample.

Claims (114)

1. Method for depth-resolved optical detection of a sample, with a scanning movement from an on or in
Illumination light distribution of at least one wavelength generated over a sample or at least part of the sample and detection, in particular of the light influenced due to interaction with the sample, in particular fluorescent light and / or reflected light and / or luminescent light and / or scattered and / or transmitted light
wherein the illuminating light has a modulation in at least one spatial direction
and the scanning movement and the detection associated with the scanning movement take place at least in the case of a first and a second different phase position of the modulation and / or first or second frequency of the periodicity of the modulation and at least one optical sectional image is calculated through the sample / the sample part. 2. The method of claim 1, wherein the modulation by impressing at least one at least one-dimensional spatial periodic structure takes place. 3. The method according to claim 1 or 2, where the light distribution is mapped onto the sample. 4. The method according to at least one of the preceding claims, wherein the sectional image is depicted. 5. The method according to at least one of the preceding claims, wherein a periodic structure is visualized. 6. The method according to at least one of the preceding claims, wherein at least one interference pattern of the sample is impressed.   7. The method according to at least one of the preceding claims, wherein several frequencies and phases of the structure are tested simultaneously be mapped. 8. The method according to at least one of the preceding claims, wherein several frequencies and / or phase positions of the structure are tested sequentially be mapped. 9. The method according to at least one of the preceding claims, wherein the lighting is provided by at least one linear light distribution, which in in the narrow direction up to a diffraction limited extent and in the another, at right angles to this, has a multiple of this extent. 10. The method according to at least one of the preceding claims, wherein the scanning is carried out point by point by means of the scanning movement. 11. The method according to at least one of the preceding claims, wherein a scan movement of a line pattern and / or within a scan field at least one one-dimensional or two-dimensional dot pattern takes place. 12. The method according to claim 11, being a composite image from the scanned line and / or Dot patterns is generated. 13. The method of claim 11 or 12, wherein an equidistant grid of line and / or point patterns is used. 14. The method according to at least one of the preceding claims, wherein the illuminating light contains several wavelengths. 15. The method according to at least one of the preceding claims, wherein at n wavelengths at least n + 1 phase positions of the structure are detected.   16. The method according to at least one of the preceding claims, wherein Phase and / or frequency for each wavelength to generate coding values is set differently. 17. The method according to at least one of the preceding claims, wherein a wavelength-dependent phase coding and / or frequency coding of the Illumination light takes place and for each wavelength using the coding optical sectional image is calculated. 18. The method according to at least one of the preceding claims, wherein the scanning process is carried out with a line with several wavelengths. 19. The method according to at least one of the preceding claims, wherein the scanning process with several lines takes place simultaneously. 20. The method of claim 19, wherein is illuminated with one or more wavelengths.   21. The method according to at least one of the preceding claims, wherein when calculating phase images (I _j (x) with the image phase: ϕ _i ) with phase coding of the excitation wavelength (ϕ _j ) images are calculated which are characteristic of the respective jth wavelength Image information from the pseudo-confocal section (C _j ) and from the background (B _j ) contain:
in which
with c _ij (x) = cos (kx + ϕ _j + ϕ _i ) - cos (kx + ϕ _j ) 22. The method according to at least one of the preceding claims, under Use a periodic structure whose period is at least in one The spatial direction (Y) changes perpendicular to the direction of the periodicity (X). 23. The method according to at least one of the preceding claims, under Use of an optical arrangement with a dispersive unit that Splitting illuminating light into spectral components and bringing them back together, the imaging optics for imaging the spectral components in a focal plane having a periodic structure in or near the focal plane wavelength-dependent influencing of the illuminating light is provided. 24. The method according to at least one of the preceding claims, wherein the generation of optical sectional images with the help of structured lighting by recording individual images with different modulation frequencies and / or image phase takes place. 25. The method according to at least one of the preceding claims, wherein the optical section thickness is varied by changing the modulation frequency. 26. The method according to at least one of the preceding claims, wherein phase coding with simultaneous imaging of different wavelengths and / or line foci on a common detector. 27. The method according to at least one of the preceding claims, wherein a Frequency coding with simultaneous imaging of different wavelengths and / or Line focus is done on a common detector. 28. The method according to at least one of the preceding claims, wherein multiple wavelengths are imaged simultaneously on the sample.   29. The method according to claim 28, where the mapping is spatially separated or on a common location of the sample he follows. 30. The method according to at least one of the preceding claims, wherein a wavelength is imaged on the sample. 31. The method according to claim 30, wherein the image is spatially separated multiple times or at a place of rehearsal. 32. The method according to at least one of the preceding claims, wherein the light distribution on the sample is linear or point-shaped. 33. The method according to at least one of the preceding claims, wherein the individual images are recorded sequentially. 34. The method according to at least one of the preceding claims, wherein multiple lines and / or point-shaped light distributions are generated. 35. The method according to at least one of the preceding claims, wherein a parallel recording of individual images takes place. 36. The method according to at least one of the preceding claims, wherein at several wavelengths, the section thicknesses by changing the respective Modulation frequency are preferably set the same. 37. The method according to at least one of the preceding claims, wherein detection with point detector and / or line detector and / or matrix detector he follows.   38. The method according to at least one of the preceding claims, wherein a wavelength-dependent phase coding by a Arrangement for generating a wavelength-dependent parallel offset along periodic structure. 39. The method according to at least one of the preceding claims, wherein a wavelength-dependent phase coding by a tilted plate in an intermediate image. 40. The method according to at least one of the preceding claims, wherein a wavelength-dependent phase coding by a dispersive element takes place. 41. The method according to at least one of the preceding claims, wherein a wavelength-dependent phase coding by a optical arrangement with a dispersive unit which the illuminating light in splitting spectral components and bringing them back together, one Has imaging optics for imaging the spectral components in a focal plane, wherein in or near the focal plane a periodic structure for wavelength-dependent influencing of the illuminating light is provided the structure being rotatable about the optical axis. 42. The method of claim 41, wherein the spectral splitting in one Intermediate picture takes place.   43. The method according to at least one of the preceding claims, wherein a frequency coding of several wavelengths by means of an optical arrangement with a dispersive unit which splits the illumination light into spectral components and merges them again, which has imaging optics for imaging the spectral components in a focal plane, in or a periodic structure is provided in the vicinity of the focal plane for influencing the illuminating light as a function of the wavelength, with
A periodic structure whose period is at least in a spatial direction (Y) perpendicular to the
Direction of periodicity (X) changes. 44. The method according to at least one of the preceding claims, wherein a Frequency coding of several wavelengths is done by the imaging scale is changed depending on the wavelength. 45. The method according to at least one of the preceding claims, wherein a Frequency coding of several wavelengths with an optical arrangement with one dispersive unit that splits the illuminating light into spectral components and merges again, the imaging optics for imaging the spectral Has portions in a focal plane, one in or near the focal plane periodic structure for influencing the wavelength dependent Illumination light is provided with a composed of several parts of different periodicity Structure takes place.   46. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images takes place with changed image phase. 47. The method of claim 46, wherein shifting the periodic structure perpendicular to the optical axis. 48. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with changed image phase A scanner is moved. 49. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with changed image phase by tilting a plane-parallel plate. 50. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with a different modulation frequency he follows. 51. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with a different modulation frequency by changing the magnification depending on the wavelength. 52. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with a different modulation frequency by swiveling in different structures with different periodicity he follows. 53. The method according to at least one of the preceding claims, wherein a sequential recording of the individual images with a different modulation frequency by shifting a periodic structure whose period is at least in a spatial direction (Y) substantially perpendicular to Direction of periodicity (X) changes, perpendicular to the periodicity.   54. The method according to at least one of the preceding claims, wherein multiple light distributions are generated on the sample and one parallel The individual images are recorded. 55. The method according to claim 54, with the generation of a plurality of light distributions on the sample at a wavelength, in particular by
Splitting the lighting into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure that acts as a beam splitter. 56. The method according to claim 54, producing multiple light distributions on the sample at multiple wavelengths, in particular by
Splitting the lighting into several partial beams with a dispersive element or
Splitting the lighting into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure that acts as a beam splitter. 57. The method according to at least one of the preceding claims, wherein a parallel recording of the individual images at different phases of the Illumination takes place. 58. The method according to claim 57 at several wavelengths, in particular by means of one
Arrangement with a dispersive element and a periodic structure which can be rotated about the optical axis, the dispersive element preferably being arranged in the intermediate image and / or one
Arrangement with a dispersive element and a periodic structure from several parts of different periodicity and the dispersive element is preferably arranged in the intermediate image and / or
Arrangement with a dispersive element, the periodic structure being reflective and acting as a beam splitter. 59. The method of claim 57 at a wavelength, in particular by means of a
Splitting the lighting into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflecting periodic structure, which acts as a beam splitter, the structure being rotatable or by means of the optical axis
Splitting the lighting into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure, which acts as a beam splitter, the structure being composed of several parts. 60. The method according to at least one of the preceding claims, wherein a parallel recording with different modulation frequency. 61. The method according to claim 60 at several wavelengths, in particular by means of
Arrangement of a dispersive element and a periodic structure, the period of which changes at least in one spatial direction (Y) substantially perpendicular to the direction of the periodicity (X) or one
Arrangement with a dispersive element, the periodic structure consisting of parts of different periodicity, the dispersive element preferably being arranged in an intermediate image. 62. The method according to claim 60 at a wavelength, in particular by means of
Splitting the lighting into several partial beams with a beam splitter AO consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure,
with a periodic structure, the period of which changes at least in one spatial direction (Y) substantially perpendicular to the direction of the periodicity (X) or
Splitting the lighting into several partial beams with a beam splitter AO consisting of at least one full mirror and a partially transparent mirror or splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure, the periodic structure consisting of parts of different periodicity. 63. Arrangement for depth-resolved optical detection of a sample, in particular containing the light of an illuminating light distribution influenced by interaction with a sample, in particular fluorescent light and / or reflected light and / or luminescent light and / or containing scattered and / or transmitted light
Means for illuminating the sample with at least one wavelength
Means for generating a relative movement between the sample and the illuminating light
Means for imaging the light influenced by the sample on at least one detector
Means for mapping an at least one-dimensionally spatially periodically changing structure onto the sample in different phases and / or frequencies of the periodicity
Means for calculating at least one optical sectional image from the location information of the light influenced by the sample. 64. Arrangement according to at least one of the preceding claims, wherein Means for the pictorial representation of the sectional image are provided. 65. Arrangement according to at least one of the preceding claims, wherein Means for imaging at least one interference pattern are provided. 66. Arrangement according to at least one of the preceding claims, wherein the lighting is provided by at least one linear light distribution, which in in the narrow direction up to a diffraction limited extent and in the another, at right angles to this, has a multiple of this extent. 67. Arrangement according to at least one of the preceding claims, wherein the scanning is carried out point by point by means of the scanning movement. 68. Arrangement according to at least one of the preceding claims, wherein a scan movement of a line pattern and / or within a scan field at least one one-dimensional or two-dimensional dot pattern takes place.   69. Arrangement according to at least one of the preceding claims, wherein a composite image from the scanned line and / or dot patterns is produced. 70. Arrangement according to at least one of the preceding claims, wherein an equidistant grid of line and / or point patterns is used. 71. Arrangement according to at least one of the preceding claims, wherein the illuminating light contains several wavelengths. 72. Arrangement according to at least one of the preceding claims, wherein the scanning process is carried out with a line with several wavelengths. 73. Arrangement according to at least one of the preceding claims, wherein the scanning process with several lines takes place simultaneously. 74. Arrangement according to at least one of the preceding claims, wherein is scanned with one or more wavelengths. 75. Arrangement according to one of the preceding claims, with a periodic Structure whose period is at least in a spatial direction (Y) perpendicular to the Direction of periodicity (X) changes. 76. Arrangement according to one of the preceding claims, with an optical Arrangement with a dispersive unit that transforms the illuminating light into spectral Splits and merges components that create an imaging optic Mapping of the spectral components in a focal plane, being in or in the A periodic structure near the focal plane for wavelength-dependent Influencing the illuminating light is provided.   77. Arrangement according to claim 76, wherein the dispersive splitting in one Intermediate image level takes place. 78. Arrangement according to one of the preceding claims, wherein phase coding with simultaneous imaging of different wavelengths and 1 or line foci on a common detector. 79. Arrangement according to one of the preceding claims, wherein one Frequency coding with simultaneous imaging of different wavelengths and / or Line focus is done on a common detector. 80. Arrangement according to one of the preceding claims wherein multiple wavelengths are imaged simultaneously on the sample. 81. Arrangement according to one of the preceding claims where the mapping is spatially separated or on a common location of the sample he follows. 82. Arrangement according to one of the preceding claims, wherein a wavelength is imaged on the sample. 83. Arrangement according to one of the preceding claims where the imaging takes place several times separately or on one location of the sample. 84. Arrangement according to one of the preceding claims, wherein the light distribution on the sample is linear or point-shaped. 85. Arrangement according to one of the preceding claims whereby the individual images are recorded sequentially.   86. Arrangement according to one of the preceding claims wherein several lines and / or point-shaped light distributions are provided. 87. Arrangement according to one of the preceding claims whereby the individual images are recorded in parallel. 88. Arrangement according to one of the preceding claims wherein the detection with point detector and / or line detector and / or Matrix detector takes place. 89. Arrangement according to one of the preceding claims for wavelength-dependent Phase encoding. 90. Arrangement according to claim 89 for generating a wavelength-dependent Parallel offset along the periodic structure. 91. Arrangement according to claim 90, with a tilted plane plate in an intermediate picture. 92. Arrangement according to claim 90 with a dispersive in the direction of the periodicity Element in an illustration pupil. 93. Arrangement for phase coding, with a optical arrangement with a dispersive unit which the illuminating light in splitting spectral components and bringing them back together, one Has imaging optics for imaging the spectral components in a focal plane, wherein in or near the focal plane a periodic structure for wavelength-dependent influencing of the illuminating light is provided the structure being rotatable about the optical axis. 94. Arrangement according to claim 93, wherein the spectral splitting in one Intermediate picture takes place.   95. Arrangement according to one of the preceding claims Frequency coding of several wavelengths with one Optical arrangement with a dispersive unit that the illuminating light in splitting spectral components and bringing them back together, one Has imaging optics for imaging the spectral components in a focal plane, wherein in or near the focal plane a periodic structure for wavelength-dependent influencing of the illuminating light is provided with a periodic structure, the period of which is at least in one spatial direction (Y) changes perpendicular to the direction of periodicity (X). 96. Arrangement according to one of the preceding claims Frequency coding of several wavelengths, where the imaging scale is changed depending on the wavelength. 97. Arrangement according to one of the preceding claims Frequency coding of several wavelengths with one optical arrangement with a dispersive unit which the illuminating light in splitting spectral components and bringing them back together, one Has imaging optics for imaging the spectral components in a focal plane, wherein in or near the focal plane a periodic structure for wavelength-dependent influencing of the illuminating light is provided with a composed of several parts of different periodicity Structure. 98. Arrangement according to one of the preceding claims sequential recording of the individual images with changed image phase while shifting the structure (PE) perpendicular to the optical axis. 99. Arrangement according to one of the preceding claims sequential recording of the individual images with changed image phase by adjusting the position of a scanner.   100. Arrangement according to one of the preceding claims sequential recording of the individual images with changed image phase by tilting a plane-parallel plate. 101. Arrangement according to one of the preceding claims sequential recording of the individual images with a changed modulation frequency Means for changing the magnification depending on the wavelength. 102. Arrangement according to one of the preceding claims sequential recording of the individual images with a changed modulation frequency Swinging in different grids with different periodicity. 103. Arrangement according to one of the preceding claims sequential recording of the individual images with changed modulation frequency by shifting a periodic structure whose period is at least in a spatial direction (Y) substantially perpendicular to Direction of periodicity (X) changes perpendicular to the periodicity. 104. Arrangement according to one of the preceding claims Generation of multiple light distributions on the sample and parallel recording of the Frames. 105. Arrangement according to one of the preceding claims Generation of multiple light distributions on the sample and parallel recording of the Single images at a wavelength in particular below Splitting the lighting into several partial beams with a beam splitter arrangement of at least one full mirror and one partially transparent mirror and / or splitting the lighting into several partial beams with at least a mirror and a reflective periodic structure that acts as a beam splitter acts. 106. Arrangement according to one of the preceding claims for generating a plurality of light distributions on the sample and parallel recording of the individual images at a plurality of wavelengths, in particular under
Splitting the lighting into several partial beams with a dispersive element and / or
Splitting the lighting into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure that acts as a beam splitter. 107. Arrangement according to one of the preceding claims parallel recording of the individual images with different phases of the Lighting. 108. Arrangement according to one of the preceding claims
parallel recording of the individual images with different phases of the lighting
at several wavelengths, in particular with one
Arrangement with a dispersive element, the periodic structure being rotatable about the optical axis and that
dispersive element is preferably arranged in an intermediate image and / or an arrangement with a dispersive element, the periodic structure being composed of several parts of different periodicity and / or
Arrangement with a dispersive element, the periodic structure being reflective and acting as a beam splitter. 109. Arrangement according to one of the preceding claims
parallel recording of the individual images with different phase positions
at a wavelength especially below
Splitting the humidification into several partial beams with a beam splitter arrangement consisting of at least one full mirror and a partially transparent mirror and / or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure, the structure being rotatable about the optical axis and / or
Splitting the lighting into several partial beams with a beam splitter AO consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflective periodic structure,
the structure being composed of several parts. 110. Arrangement according to one of the preceding claims parallel recording with different modulation frequency. 111. Arrangement according to one of the preceding claims for parallel recording with different modulation frequency at several wavelengths, in particular with one
Arrangement with a dispersive element and a periodic structure, the period of which changes at least in one spatial direction (Y) substantially perpendicular to the direction of the periodicity (X) and / or
Arrangement with a dispersive element and a periodic structure, the periodic structure consisting of parts of different periodicity,
wherein the dispersive element is preferably arranged in the intermediate image. 112. Arrangement according to one of the preceding claims for parallel recording with different modulation frequency at a wavelength in particular below
Splitting the lighting into several partial beams with a beam splitter AO consisting of at least one full mirror and a partially transparent mirror and / or
Splitting the lighting into several partial beams with at least one mirror and a reflecting periodic structure, which acts as a beam splitter and / or with a periodic structure, the period of which at least in one spatial direction (Y) is substantially perpendicular to the
Direction of periodicity (X) changes and / or
Splitting the lighting into several partial beams with a beam splitter AO consisting of at least one full mirror and a partially transparent mirror or
Splitting the lighting into several partial beams with at least one mirror and a reflecting periodic structure, which acts as a beam splitter, the periodic structure consisting of parts of different periodicity. 113. Arrangement according to at least one of the preceding claims, wherein before the Detector arrangement at least one dispersing element is provided. 114. Arrangement according to at least one of the preceding claims in a microscope, preferably a laser scanning microscope.