DE19859314A1 - Light diffraction device for separating excitation and emission light in confocal microscope e.g. laser scanning microscope, uses at least one diffraction element for diffraction of selected wavelength of excitation light - Google Patents

Abstract

The light diffraction device uses at least one light diffraction element, e.g. an acousto-optical tunable filter (AOTF), inserted in the light path of both the excitation and the emission beams in a confocal microscope, for selective diffraction of at least one selected wavelength of the excitation beam, the emission light passing through the diffraction element without being affected.

Description

In Fig. 1-3 arrangements by way of example the invention will be explained. In Fig. 1, the light (excitation light) of two lasers L1, L2 with different wavelengths is coupled into a common beam path via a mirror SP and a beam splitter ST, which on the side S1 of a mirrored prism in the direction of an AOTF (Acousto-Optical Tunable Filter ) is reflected. The excitation light is introduced into the AOTF, with light diffracted in the first order for the wavelength set via the control frequency of the AOTF being deflected exactly in the direction of a pinhole PH with upstream and downstream pinhole optics PHO for setting the beam profile, while other possible wavelengths are undeflected in zero Cross the AOTF and don't get to the pinhole.

The pinhole PH serves here as an excitation and detection pinhole at the same time. The excitation light is generated via scanning units SC1, SC2 and a scanning optics SCO in the direction of a microscopic beam path MI in the direction of a sample pictured.

The light emitted by the sample, consisting of parts of the excitation light and wavelength shifted fluorescence components, passes through the light path in reverse direction to the AOTF. This is where the wavelength components of the Excitation light in turn via first-order diffraction on the mirror side S1 of the prism PS, while the fluorescence components undiffracted the AOTF in zero Cross order and thereby an angle to the reflected excitation light take in.

Between the returning zeroth and first order rays is exactly that Point arranged between the prism surfaces S1 and S2, whereby the Fluorescent light hits side S2 and from it towards one Detection unit, here consisting for example of a line filter LF, a Color splitter NFT and two detectors for different wavelengths, reflected becomes.  

Due to the low bandwidth of the AOTF of approx. 2 nm bandwidth for the Excitation light acts as an extreme edge filter with clear advantages, for example against dichroic filters with bandwidths greater than 10 nm.

This is particularly important because the distance from the excitation wavelength and fluorescence wavelengths can be less than 10 nm and through which arrangement according to the invention nevertheless a wavelength-dependent separation is possible.

By changing the frequency, the AOTF can change from the wavelength of the laser L1 the wavelength of the laser L2 are switched and again that Excitation light can be separated from fluorescent light.

Instead of the prism with sides S1, S2, two independent sides can be used S1, S2 corresponding but not connected mirrors are used. An advantage is that they can also be rotated to provide an accurate To enable adjustment to the AOTF or the detection DE.

In Fig. 2 is a similar arrangement with only a scanner SC is illustrated. Here, instead of the prism, a mirror S is provided, which deflects the excitation light in the direction of the AOTF analogously to FIG. 1, the fluorescence light returning in zero order here passing through the AOTF next to the mirror S and in this way in the direction of one here detection, not shown.

In principle, arrangements are also conceivable in which the AOTF alone as Separation unit of excitation light and fluorescent light can serve by the Laser light in the direction of the first order without an upstream element in the AOTF arrives and the detection light at an angle to the excitation light AOTF leaves and goes directly into a detection unit, which is just Has an impact on the overall length, as the angle is, for example, four degrees turns out quite small and overlaps of the wavelength components are avoided should.

Furthermore, a separating mirror can also be used only for the fluorescent light be provided.

In Fig. 3, a further advantageous embodiment in the form of a non-mirrored prism is provided inside through refraction, the light of an excitation laser to first order in the AOTF and deflects the zeroth order (the fluorescent light) in the direction of the detection DE.

By the angle between first and zero order and different Wavelengths is advantageously a clear separation of the wavelength components possible.

The invention is particularly advantageous in using a laser scanning microscope applicable to an AOTF.

Other advantageous applications of another light diffractive element Radiation separation through different diffraction orders are however in one microscopic beam path conceivable and advantageous in the scope of the invention locked in.

Claims (7)

1. Arrangement of a light diffractive element for the separation of excitation and Emission light in a microscopic beam path, preferably in one confocal microscope, and especially in a laser scanning microscope. 2. Arrangement according to claim 1, wherein the light diffractive element from both Excitation light and emission light are passed through. 3. Arrangement according to one of the preceding claims, wherein the light diffractive element has at least one wavelength of excitation affected by diffraction while other wavelengths emitted by the sample pass through the element unaffected and thereby spatially by the excitation light be separated. 4. Arrangement according to one of the preceding claims, being in the excitation beam path in front of the element and / or in Detection beam path after the element for improved separation of the Light components at least one optical element influencing the direction of light is provided. 5. Arrangement according to one of the preceding claims, with an AOTF as a light diffractive element. 6. Arrangement according to one of the preceding claims, with a reflection element as an optical element 7. Arrangement according to one of the preceding claims, with a refractive element as an optical element.