HK1038797B - Arrangement for separation excitation light and emission light in a microscope - Google Patents

Description

The present invention relates to a fluorescence microscope and a device for coupling light into a beam of a microscope.

Priority older publication WO 99/42884 describes an optical arrangement in which a laser beam from one or more lasers is coupled to a microscope, in particular a confocal fluorescence laser scanning microscope, using an AOTF or AOD as an acoustical element in the first order of refraction.

The latter is used to selectively control individual wavelengths in their power after beam merging.

The subject of US 4.627.730 is an electronically grating microscope, which uses a Bragg cell as an acoustical element to split the excitation light into a beam to be grated and a reference beam.

EP 0 503 236 A2 describes a device and a procedure for the microscopic examination of samples, particularly of semiconductor chips, which allows a sample with a particularly large numerical aperture and thus a particularly good resolution to be examined.

US 5.751.417 refers to a confocal fluorescence microscopy device which uses multiple prisms as dispersive elements and a sliding grid to select desired wavelength ranges for detection in the beam path.

EP 0 562 488 A1 describes a device for high-resolution scanning, such as a laser raster microscope, a barcode scanner or a laser printer, which uses a holographic grid in the beam path to increase the resolution, through which both the excited and the reflected light pass.

EP 0 620 458 A1 refers to an optical waveguide made of a material having an anisotropy of the refractive index.

JP-04157413 shows a scanning microscope with an acousto-optical modulator in the beam direction to control the intensity of excitation.

WO 97/30371 concerns a light microscope which uses a first AOTF to separate a beam of light into two beams of different polarization.

EP 0 327 425 A1 describes a confocal optical microscopy procedure and device which uses several lasers of different wavelengths to obtain three-dimensional microscopic images more quickly, modulating the wavelengths by means of acousto-optical modulators and coupled to a beam divider.

The subject of JP-01282515 is a raster microscope in which the light from a number of lasers of different wavelengths is coupled into a microscope beam path. Each colour can be modulated by an acoustic optical modulator before entering the beam path.

The subject of DE 196 33 185 A1 is a point light source for a laser scanning microscope and a method for coupling at least two lasers of different wavelengths into a laser scanning microscope.

DE 197 02 753 A1 concerns a laser scanning microscope which uses an AOTF to couple radiation into a light conductor.

DE 196 27 568 A1 describes an arrangement and procedure for confocal microscopy, whereby the light of a number of lasers is coupled to a microscope beam with a diffraction grating.

EP 0 148 803 is a device for splitting a multicolored beam into a number of parallel monochrome beams, using several acoustic optical elements at different frequencies.

The purpose of the invention is to create a fluorescent microscope and a device for coupling light into a beam path of a microscope, which allows improved introduction of light into the microscopic beam path and improved separation of excitation light and wavelength-shifted emission light.

This problem is solved in a first aspect of the invention by the fluorescence microscope with the features of claim 1.

In other aspects of the invention, this task is solved by the fluorescence microscope with the features of claim 5, by the fluorescence microscope with the features of claim 7.

Figures 1-3 illustrate the design of the devices according to the invention. In Fig. 1 the light (stimulation light) of two lasers L1,L2 of different wavelengths is coupled to a common beam path by means of a mirror SP and a beam splitter ST, which is reflected at the side S1 of a mirrored prism in the direction of an AOTF (Acousto-OpticalTunable Filter). The excitation light is introduced into the AOTF, whereby first order wavelength-curved light set above the control frequency of the AOTF is deflected exactly in the direction of a pinhole PH with pre- and secondary pinhole optics PHO to adjust the beam profile, while other possible wavelengths uncurved in order zero pass through the AOTF And not get to the pinhole.Other The PH pinhole serves as both the stimulation and detection pinhole. The test chemical is then applied to the sample in the direction of a microscopic beam of MI. The light emitted by the sample consisting of parts of the excitation light The light path passes through the optical path in the opposite direction to the AOTF, where the wavelength proportions of the excitation light reach the mirror side S1 of the prism PS via the first-order deflection, while the fluorescence proportions pass through the AOTF unfolded in the zero-order direction, thus taking an angle to the reflected excitation light. Between the zero and first order returning rays, the tip is now exactly placed between the prism surfaces S1 and S2,whereby the fluorescent light is reflected from side S2 towards a detection unit, for example consisting of a line filter LF, a colour divider NFT and two detectors for different wavelengths.

The low bandwidth of the AOTF of about 2 nm for the excitation light makes it an extreme edge filter with clear advantages, for example, over dichroic filters with bandwidths greater than 10 nm. This is of particular importance because the distance between excitation wavelengths and fluorescence wavelengths may be less than 10 nm and the arrangement of the invention nevertheless allows a wavelength-dependent separation. By changing the frequency, the AOTF can be switched from the laser wavelength L1 to the laser wavelength L2 and the excitation light can be separated from the fluorescent light.

Instead of the prism with sides S1, S2, two independent mirrors corresponding to the sides S1, S2 but not connected may also be used. One advantage is that they can also be rotated to allow precise adjustment to the AOTF or detection of DE. Figure 2 shows a similar arrangement with only one scanner SC, where instead of the prism a mirror S is provided which redirects the excitation light in the direction of the AOTF analogously to Figure 1, whereby the fluorescent light passing through the AOTF passes in the direction of the mirror S and thus in the direction of a detection not shown here.

In principle, arrangements are also conceivable in which the AOTF can serve as the only separation unit of the excitation light and the fluorescent light by the laser light entering the AOTF in the first order direction without a forward element and the detection light leaving the AOTF at an angle to the excitation light and entering directly into a detection unit, which has only an effect on the length of the structure, since the angle is quite small, for example, four degrees and the aim is to avoid overlapping of the wavelength ratios. Furthermore, a separate mirror may be provided for the fluorescent lamp only. Figure 3 shows another advantageous design in the form of an unspectacled prism which, by refraction, first absorbs the light of an excitation laser. The detector shall be capable of detecting the emission of any of the following: The angle between first and zero order and different wavelengths makes it possible to separate the wavelength ratios. The invention is particularly useful in a laser scanning microscope with an AOTF. However, other advantageous applications of another refracting element for separating radiation by different refractive orders are conceivable in a microscopic beam path and are advantageously included in the scope of the invention. This can be used to regulate the stimulation intensity.

In Fig. 4 several such elements are advantageously provided, here AOTF and AOM in the laser beam gate to couple the laser radiation.

Claims (17)

1. Fluorescence microscope, in particular confocal fluorescence laser microscope, comprising a radiation source (L₁, L₂, L₃), in particular a laser which emits excitation light for a sample, a detection device (DE, DT, NFT) for detecting emission light emitted by the sample, microscope optics for directing excitation light onto the sample and for directing emission light back towards the radiation source and the detection device, an acousto-optical element (AOM, AOTF) for diffracting excitation light, by means of which an intensity of the diffracted excitation light is regulated and which is arranged between the radiation source and the microscope optics such that diffracted excitation light is introduced into the microscope optics (SC1, SC2, SCO, M1),

   - wherein the emission light emitted by the sample includes fractions of excitation light and fractions of wavelength-shifted fluorescence light,

   - excitation light emitted by the sample is deflected by the acousto-optical device (AOM, AOTF) through diffraction towards the radiation source,

   - and wavelength-shifted fluorescence light emitted by the sample is transmitted in an undiffracted manner through the acousto-optical element (AOM, AOTF) and is spatially separated from the excitation light fractions of the emission light and

   - wherein the detection device (DE, DT, NFT) is arranged with respect to the acousto-optical element such that wavelength-shifted fluorescence light transmitted in an undiffracted manner through the acousto-optical element (AOM, AOTF) is detected by means of the detection device (DE, DT, NFT), and

   comprising a filter device (LF) which is arranged between the acousto-optical element and the detection device (DE, DT, NFT) for selectively detecting wavelength-shifted fluorescence light in the detection device (DE, DT, NFT).
2. Fluorescence microscope according to claim 1, characterized in that for an improved separation of the light fractions at least one optical element influencing the direction of light is provided in an excitation beam path before the acousto-optical element (AOM, AOTF) and/or in a detection beam path after the acousto-optical element (AOM, AOTF).
3. Fluorescence microscope according to claim 2, characterized in that as optical element a reflection element (S1, S2, PS, S), in particular a mirror (S), a double mirror (S1, S2) or a vaporized prism (PS) is provided.
4. Fluorescence microscope according to any one of claims 2 or 3, characterized in that as optical element or as further optical element a refractive element (P), in particular a non-vaporized prism (P) is provided in an excitation beam path before the acousto-optical element (AOM, AOTF) and/or in a detection beam path after the acousto-optical element (AOM, AOTF).
5. Fluorescence microscope, in particular confocal fluorescence laser microscope, comprising a radiation source (L₁, L₂, L₃), in particular a laser which emits excitation light for a sample, a detection device (DE, DT, NFT) for detecting emission light emitted by the sample, microscope optics for directing excitation light onto the sample and for directing emission light back towards the radiation source and the detection device, an acousto-optical element (AOM, AOTF) for diffracting excitation light, which is arranged between the radiation source and the microscope optics such that diffracted excitation light is introduced into the microscope optics (SC1, SC2, SCO, M1),

   - wherein the emission light emitted by the sample includes fractions of excitation light and fractions of wavelength-shifted fluorescence light,

   - excitation light emitted by the sample is deflected by the acousto-optical device (AOM, AOTF) through diffraction towards the radiation source,

   - and wavelength-shifted fluorescence light emitted by the sample is transmitted in an undiffracted manner through the acousto-optical element (AOM, AOTF) and is spatially separated from the excitation light fractions of the emission light and

   - wherein the detection device (DE, DT, NFT) is arranged with respect to the acousto-optical element such that wavelength-shifted fluorescence light transmitted in an undiffracted manner through the acousto-optical element (AOM, AOTF) is detected by means of the detection device (DE, DT, NFT),

   comprising a filter device (LF) which is arranged between the acousto-optical element and the detection device (DE, DT, NFT) for selectively detecting wavelength-shifted fluorescence light in the detection device (DE, DT, NFT), and at least one refractive element (P), in particular a non-vaporized prism (P), for influencing the direction of light and for separating the light fractions, which is arranged in an excitation beam path before the acousto-optical element (AOM, AOTF) and/or in a detection beam path after the acousto-optical element (AOM, AOTF).
6. Fluorescence microscope according to any one of claims 1 to 5, characterized in that in the direction of the microscope optics (SC1, SC2, SCO, M1) first AOM and subsequently AOTF are provided as acousto-optical elements (AOM, AOTF) .
7. Fluorescence microscope, in particular confocal fluorescence laser microscope, comprising a radiation source (L₁, L₂, L₃), in particular a laser which emits excitation light for a sample, a detection device (DE, DT, NFT) for detecting emission light emitted by the sample, microscope optics for directing excitation light onto the sample and for directing emission light back towards the radiation source and the detection device, a plurality of acousto-optical elements (AOM, AOTF) for diffracting excitation light, which are arranged between the radiation source and the microscope optics such that diffracted excitation light is introduced into the microscope optics (SC1, SC2, SCO, M1),

   - wherein in the direction of the microscope optics (SC1, SC2, SCO, M1) first AOM and subsequently AOTF are arranged as acousto-optical elements (AOM, AOTF),

   - the emission light emitted by the sample includes fractions of excitation light and fractions of wavelength-shifted fluorescence light,

   - excitation light emitted by the sample is deflected by the acousto-optical devices (AOM, AOTF) through diffraction towards the radiation source,

   - wavelength-shifted fluorescence light emitted by the sample is transmitted in an undiffracted manner through the acousto-optical elements (AOM, AOTF) and is spatially separated from the excitation light parts of the emission light and

   - wherein the detection device (DE, DT, NFT) is arranged with respect to the acousto-optical elements such that wavelength-shifted fluorescence light transmitted in an undiffracted manner through the acousto-optical elements (AOM, AOTF) is detected by means of the detection device (DE, DT, NFT), and

   comprising a filter device (LF) which is arranged between the acousto-optical elements and the detection device (DE, DT, NFT) for selectively detecting wavelength-shifted fluorescence light in the detection device (DE, DT, NFT),

   - wherein the radiation source (L₁, L₂, L₃) is designed as a plurality of lasers (L₁, L₂, L₃) of different wavelength,

   - a plurality of acousto-optical elements (AOM, AOTF) is provided, with at least one acousto-optical element (AOM, AOTF) being assigned to each laser (L₁, L₂, L₃),

   - the different wavelengths are coupled in simultaneously or individually into a microscope beam path (SC1, SC2, SCO, M1) through diffraction in the respective acousto-optical elements (AOM, AOTF) and

   - wavelength-shifted emission light and excitation light of, in each case, a different wavelength is transmitted in an undiffracted manner through the respective acousto-optical elements (AOM, AOTF).
8. Fluorescence microscope according to any one of claims 1 to 7, characterized in that at least one glass fiber is provided for the coupling-in of the excitation light.
9. Fluorescence microscope according to any one of claims 7 or 8, characterized in that for an improved separation of the light fractions at least one optical element influencing the direction of light is provided in an excitation beam path before the acousto-optical element (AOM, AOTF) and/or in a detection beam path after the acousto-optical element (AOM, AOTF).
10. Fluorescence microscope according to any one of claims 1 to 6, characterized in that

    - the radiation source (L₁, L₂, L₃) is designed as a plurality of lasers (L₁, L₂, L₃) of different wavelength,

    - a plurality of acousto-optical elements (AOM, AOTF) is provided, with at least one acousto-optical element (AOM, AOTF) being assigned to each laser (L₁, L₂, L₃),

    - the different wavelengths are coupled in simultaneously or individually into a microscope beam path (SC1, SC2, SCO, M1) through diffraction in the respective acousto-optical elements (AOM, AOTF) and

    - wavelength-shifted emission light and excitation light of, in each case, a different wavelength is transmitted in an undiffracted manner through the respective acousto-optical elements (AOM, AOTF).
11. Fluorescence microscope according to any one of claims 1 to 10, characterized in that AOTF and/or AOM are provided as acousto-optical elements.
12. Fluorescence microscope according to claim 10, characterized in that the excitation power of each laser (L₁, L₂, L₃) can be adjusted individually with the respective acousto-optical element (AOM, AOTF).
13. Fluorescence microscope according to any one of claims 1 to 12, characterized in that the acousto-optical elements (AOM, AOTF) can be switched through a frequency change from a first wavelength of a first laser to a second wavelength of a second laser.
14. Fluorescence microscope according to any one of claims 1 to 13, characterized in that through diffraction at the acousto-optical element (AOM, AOTF) the excitation light is introduced in the first diffractive order into the microscope optics (SC1, SC2, SCO, M1).
15. Fluorescence microscope according to any one of claims 1 to 14, characterized in thata pinhole (PH) is provided as excitation and detection pinhole before the microscope optics (SC1, SC2, SCO, M1).
16. Fluorescence microscope according to any one of claims 7 to 15, characterized in that the radiation of the plurality of lasers (L₁, L₂, L₃) is coupled into the microscope beam path successively in the direction of the microscope optics (SC1, SC2, SCO, M1) in a given order of decreasing wavelength.
17. Fluorescence microscope according to any one of claims 1 to 16, characterized in that UV-light, visible light and/or infrared light are coupled into the microscope beam path.