DE102005040833A1 - TIRF-type microscope optical arrangement, has illumination light carried in beam paths outside microscope objective - Google Patents

Abstract

An optical arrangement for microscopic observation has on one side an immersion medium (8) and on the other - a cover glass (3) with florescence excited illumination light passed through the immersion medium and the cover glass onto the boundary/threshold surface (9). The illumination light (4) is carried in beam paths outside the microscope objective (1) on the basis on the diffraction produces by the diffraction grid (6) at least a part (B) of the illumination light is directed at a given angle (delta) on to the boundary surface (9) leading to total reflection at this surface.

Description

The The invention relates to an optical arrangement for microscopic Observation of a sample with TIRF illumination, comprising a microscope objective, one between the microscope objective and the sample arranged, at least partially with the sample in contact standing cover glass and on the interface between the coverslip and the specimen directed fluorescent stimulating illumination light.

When TIRF microscopy (TIRF - Total Internal reflection fluorescence) becomes a field of microscopy in which the sample located behind a cover glass is illuminated at such a shallow angle that the illumination light at the interface is totally reflected between cover glass and sample. It forms an evanescent wave field that affects the part of the sample, the is in direct contact with the coverslip. This influence can be observed conclusions are drawn on sample properties won.

The Principle of the generation of evanescent wavelengths in connection with the TIRF microscopy is exemplified in Kramer, "Evanescent Waves in Microscopy ", Journal Photonik, Issue 2/2004, page 42ff.

It Optical arrangements are known in which the TIRF illumination as epi-illumination by the microscope objective on the interface between coverslip and sample is directed. Because of the necessary flat angles, under which the Lighting light must hit the cover glass, this can only very high-aperture Microscope lenses are used, because to make sure that no light on Directly to the sample, which would disturb the TIRF illumination, is always only the outermost edge zone of the microscope objective for the illumination light available. The aperture must therefore significantly larger than Be 1.4.

Such high-aperture microscope objectives are relatively expensive. That is the reason for this, why the TIRF microscopy currently very expensive.

From Based on this prior art, the invention has the object underlying, an optical arrangement for microscopic observation to develop a specimen at TIRF lighting, the use of Enables microscope lenses with lower numerical aperture and less costly.

• – das Beleuchtungslicht, der Dunkelfeldbeleuchtung bei Mikroskopen entsprechend, außerhalb des Mikroskopobjektivs geführt ist und
• – im Beleuchtungslicht ein Beugungsgitter so angeordnet ist, daß aufgrund der Beugung mindestens ein Teil des Beleuchtungslichtes unter einem Winkel δ auf die Grenzfläche zwischen dem Deckglas und der Probe gerichtet ist, der zur Totalreflexion an der Grenzfläche führt.

This object is achieved with an optical arrangement of the type described above, in which

• - The illumination light, the dark field illumination in accordance with microscopes, outside the microscope objective is performed and
• - In the illumination light, a diffraction grating is arranged so that due to the diffraction at least a portion of the illumination light is directed at an angle δ on the interface between the coverslip and the sample, which leads to the total reflection at the interface.

This works according to the invention Illumination light no longer as usual by the microscope objective through, but is based on the principle of dark field illumination in beam paths outside guided the microscope objective. This is achieved that the Microscope lens to record only the detection beam path has, for which apertures smaller than 1.4 are sufficient.

Around nevertheless the total reflection at the interface between coverslip and sample bring about and thus to generate the required evanescent wave field, must on the one hand between the diffraction grating and the coverslip an immersion medium sufficiently high refractive index and on the other hand, the illumination light be diffracted at the diffraction grating so that at least a part of the illumination light to the interface arrives and is totally reflected there.

This is achieved, for example, when the diffraction grating has a lattice constant which causes the Illumination light of the first diffraction order at an angle δ to the cover glass and directed to the sample.

Farther it is advantageous if as a diffraction grating on a substrate trained phase grating provided and this between the microscope objective and the coverslip is positioned. The refractive indices should be of the coverslip, the immersion medium and the substrate are about the same be great. It is particularly advantageous if the difference of the refractive indices mutually equal to or less than 0.1.

mit

• – d der Strukturbreite des Phasengitters,
• – λ der Wellenlänge des Beleuchtungslichtes,
• – n₁ der Brechzahl des Substrates, auf welches das Phasengitter aufgebracht ist,
• – n₀ der Brechzahl der Probensubstanz,
• – α dem Winkel, unter dem der Beleuchtungsstrahlengang in das Substrat eintritt, auf dem das Phasengitter aufgebracht ist,
• – t der Strukturtiefe des Phasengitters unter der Voraussetzung einer Brechzahldifferenz von (n₁ – 1) im Phasengitter, und
• – s der Quadratwurzel aus n₁·n₁ – cosα·cosα.

Total reflection is achieved, for example, when the phase grating meets the following conditions:

With

• D the structure width of the phase grating,
• Λ of the wavelength of the illumination light,
• N _{1 is} the refractive index of the substrate to which the phase grating is applied,
• - n ₀ the refractive index of the sample substance,
• Α is the angle at which the illumination beam path enters the substrate on which the phase grating is applied,
• - t the structure depth of the phase grating assuming a refractive index difference of (n ₁ - 1) in the phase grating, and
• - s is the square root of n ₁ · n ₁ - cosα · cosα.

The invention will be explained in more detail with reference to an embodiment. The associated 1 shows the basic structure of the optical arrangement according to the invention.

In 1 is a microscope objective 1 symbolically represented, in which that of a sample 2 coming light enters. It is illuminated behind a cover glass 3 arranged sample 2 with illumination light 4 in a beam path outside the microscope objective 1 is guided.

On the way to the rehearsal 2 meets the illumination light 4 first on a ring mirror 5 centering to the microscope lens 1 is arranged, and from which it first on a diffraction grating 6 is reflected, in such a way that the illumination light 4 at an angle α to the diffraction grating 6 and the substrate 7 meets, on which the diffraction grating 6 , exemplified as a phase grating, is applied.

When passing through the substrate 7 becomes the illumination light 4 Broken. In this case, the angle of inclination β would be below the fraction A of the illumination light from the substrate 7 towards the sample 2 leaking, too steep, to the desired total reflection at the interface between the coverslip 3 and the sample 2 to be able to cause.

This proportion A would therefore be a direct light to the sample 2 and disturb the TIRF lighting. Due to the effect of the diffraction grating 6 however, since it corresponds to the 0th order of the illuminating light, the proportion A is canceled out. It can in this respect therefore no disturbing illumination light for the sample 2 reach.

The -1st order diffracted portion B 'of the illumination light 4 occurs at an even steeper angle of inclination γ than the fraction A from the substrate 7 Therefore, it is also not suitable for generating total reflection, but it runs on the sample 2 and therefore can not disturb the TIRF lighting.

On the other hand, the portion B of the illuminating light diffracted to the 1st order undergoes exiting from the substrate 7 a significant flattening and reached after passing through an immersion medium 8th and the coverslip 3 the interface 9 between sample 2 and cover glass 3 at an angle δ as desired for total reflection at this interface 9 leads and thereby forms an evanescent wave field, which is the part of the sample 2 which is in direct contact with the coverslip 3 stands.

With this arrangement is the observation of the sample in TIRF illumination with microscope objectives possible, whose aperture can be much smaller than has been required in the prior art. The object underlying the invention is achieved.

• – die Wellenlänge λ des Beleuchtungslichts 546 nm,
• – die Brechzahl des Substrates 9 n₁ = 1.52,
• – die Brechzahl der Probensubstanz n₀ = 1.33,
• – die Brechzahl des Immersionsmediums n = 1,53 bis 1,4
• – der Winkel α = 10°.

In a concrete embodiment,

• The wavelength λ of the illumination light 546 nm,
• The refractive index of the substrate 9 n ₁ = 1.52,
• The refractive index of the sample substance n ₀ = 1.33,
• - The refractive index of the immersion medium n = 1.53 to 1.4
• - the angle α = 10 °.

When Immersion medium, for example, an immersion oil with the Refractive index n = 1.518 can be used.

• – eine notwendige Strukturbreite d des Phasengitters mit d = 1,2 μm,
• – s = 1,185
• – eine erforderliche Strukturtiefe t des Phasengitters mit t = 72 nm unter der Voraussetzung einer Brechzahldifferenz von (n₁ – 1) im Phasengitter.

Under these conditions arise

• A necessary structure width d of the phase grating with d = 1.2 μm,
• - s = 1.185
• - A required structure depth t of the phase grating with t = 72 nm, assuming a refractive index difference of (n ₁ - 1) in the phase grating.

1

microscope objective

2

sample

3

cover glass

4

illumination light

5

ring mirror

6

phase grating

7

substratum

8th

Immersion medium

9

interface

A, B, B '

shares of the illumination light

Claims (8)

Optical arrangement for the microscopic observation of a sample, comprising: - a microscope objective ( 1 ), - on the one hand with an immersion medium ( 8th ) and on the other hand at least partially with the sample ( 2 ) cover glass ( 3 ), - through the immersion medium ( 8th ) and the cover glass ( 3 ) through to the interface ( 9 ) between cover glass ( 3 ) and sample ( 2 ), fluorescence-exciting illumination light ( 4 ), where - due to total internal reflection at this interface ( 9 ) forms a wave field that covers the part of the sample ( 2 ) which is in direct contact with the coverslip ( 3 ), and - from this influence on the properties of the sample ( 2 ) Obtained observation result, characterized in that - the illumination light ( 4 ) in beam paths outside the microscope objective ( 1 ), - in the illumination light ( 4 ) a diffraction grating ( 6 ), and - due to the diffraction thereby achieved at least a portion (B) of the illumination light ( 4 ) at an angle δ to the interface ( 9 ) for total reflection at the interface ( 9 ) leads. Optical arrangement according to Claim 1, in which the diffraction grating ( 6 ) causes the illumination light ( 4 ) of the first diffraction order at the angle δ on the interface ( 9 ). Optical arrangement according to Claim 1 or 2, in which the diffraction gratings ( 6 ) provided on a substrate phase grating and this between the microscope objective ( 1 ) and the cover glass ( 3 ) is arranged. Optical arrangement according to Claim 3, in which the refractive indices of the cover glass ( 3 ), of the immersion medium ( 8th ) and the substrate ( 7 ) are about the same size. An optical arrangement according to claim 4, wherein the difference the refractive indices are equal to or less than 0.1. Optical arrangement according to one of the preceding claims, in which as immersion medium ( 8th ) An immersion oil is provided. Optical arrangement according to one of the preceding claims, in which the phase grating fulfills the condition:

with - d of the structure width of the phase grating, - λ the wavelength of the illumination light, - n ₁ is the refractive index of the substrate on which the phase grating is applied, - n ₀ is the refractive index of the sample substance, - α the angle at which the illumination beam path into the substrate occurs, on which the phase grating is applied, - t the structure depth of the phase grating assuming a refractive index difference of (n ₁ - 1) in the phase grating, and - s the square root of n ₁ · n ₁ - cosα · cosα. Optical arrangement according to one of the preceding claims with the angle α <25 °.