DE102007058558A1 - Phase contrast microscope for analyzing sample, has optical unit producing reproduction beam path from light, and optical element connected with control circuit for changing phase of light during observation of sample - Google Patents

Abstract

The microscope has an optical unit for producing an illumination beam path from a light produced by a light source, where the beam path is directed by a sample. The optical unit produces a reproduction beam path from a light, which is transmitted, reflected or controlled by the sample. The reproduction beam path is directed to a receiving device. An optical arrangement is arranged in an image beam path for changing the phase of the light. An optical element (2) is connected with a control circuit (3) for changing the phase of the light during the observation of the sample.

Description

The This invention relates to a phase contrast microscope, essentially comprising: a light source, a sample to be observed, optical means for generating a directed onto the sample illumination beam path from the light coming from the light source, optical means for Generation of an imaging beam path from the transmitted from the sample, reflected or scattered light, a receiving device on which the imaging beam path is directed, and one in the imaging beam path arranged optical arrangement for changing the phase of the light.

• – Die Methode der „Quantitative Phase Microscopy", bei welcher im Hellfeldverfahren Bildinformationen von der Probe aus einer fokussierten und mindestens zwei defokussierten Mikroskopeinstellungen gewonnen werden und anschließend mit Hilfe mathematischer Methoden aus diesen Bildinformationen eine Abbildung der Probe rekonstruiert wird;
• – Holographische Verfahren auf der Basis von auf die Probe gerichtetem kohärentem Beleuchtungslicht;
• – Eine Verfahrensweise unter Verwendung eines sogenannten "Rotated Lateral Shearing Interferometer", bei dem ein C-DIC-Prisma in Rotation versetzt und während einer Umdrehung des Prismas Bildinformationen der Probe aufgenommen werden, aus denen anschließend eine Abbildung der Probe mit Hilfe mathematischer Methoden rekonstruiert wird. Das C-DIC-Prisma ist dabei in zirkular polarisiertem Licht angeordnet;
• – Das Spiralphasenkontrast-Verfahren, bei dem eine Spiralphasenmaske in die Objektivpupille oder in die Abbildungsebene der Objektivpupille eingebracht wird. In dieses Sachgebiet ist auch die nachfolgend beschriebene Erfindung einzuordnen.

For the microscopic analysis of a sample on the basis of the phase of the light influenced by the sample, essentially the following methods are known:

• - The method of "Quantitative Phase Microscopy", in which in bright field method image information from the sample from a focused and at least two defocused microscope settings are obtained and then using mathematical methods from this image information, an image of the sample is reconstructed;
• Holographic methods based on coherent illuminating light directed at the sample;
• - A procedure using a so-called "Rotated Lateral Shearing Interferometer", in which a C-DIC prism is set in rotation and recorded during a revolution of the prism image information of the sample, from which subsequently an image of the sample using mathematical methods is reconstructed , The C-DIC prism is arranged in circularly polarized light;
• - The spiral phase contrast method, in which a spiral phase mask is introduced into the objective pupil or into the imaging plane of the objective pupil. In this field, the invention described below is to be classified.

The spiral phase mask is an optical element with a phase structure Φ (r, φ) according to the function Φ (r, φ) = e ^-iφ , which extends in a spiral around its center. The center lies in the optical axis of the microscope beam path. As a result, a phase delay is imparted to the light passing through this optical element, this delay being independent of the distance of the light to the center of the optical element or to the optical axis of the microscope beam path.

The The n-th diffraction order and the -n-th order of diffraction are center-symmetric through the spiral phase mask and are doing 180 ° against each other delayed which leads to a destructive interference in the image plane.

has the sample has a phase gradient, come the nth and -nth Diffraction order, dependent of size and direction of the phase gradient, delayed against each other at the spiral phase mask and can no longer destructive in the image plane interfere.

It creates a relief-like image. Will the spiral phase mask around her Center or rotated about the optical axis of the microscope beam path, the changes Relief his direction. This effect becomes a microscopic analysis a sample used on the basis of the phase.

The Spiral phase mask is designed so that the 0th diffraction order passes through the center of the spiral phase mask and thereby is always delayed by a constant amount in the phase. The 0th diffraction order is therefore in the state of the art known spiral phase mask disadvantageously not on the sample analysis involved.

One Another disadvantage of the previously known use of a spiral phase mask is that mechanical Drive elements for generating the rotational movement about the optical Axis and high precision Rotary bearings for the spiral phase mask is required. Such mechanical functional elements are subject to wear, As a result, they are cost-intensive and often have to be time-consuming readjusted become.

From that Based on the invention, the object is an optical Arrangement for To provide a phase contrast microscope, with which the disadvantages of the prior art are eliminated and so the spiral phase contrast method with higher Efficiency executed can be.

• – ein erstes optisches Element aufweist mit einer Struktur zur Veränderung der Phase der n-ten Beugungsordnung des Lichtes, mit n einer ganzen Zahl, und
• – ein zweites optisches Element aufweist mit einer Struktur zur Veränderung der Phase der 0-ten Beugungsordnung des Lichtes, wobei

mindestens das zweite optische Element mit einer Ansteuerschaltung zur Veränderung der Phase des Lichtes während der Beobachtung der Probe verbunden ist.According to the invention this object is achieved in a phase contrast microscope of the type mentioned, by the arrangement for changing the phase of the light

• A first optical element having a structure for changing the phase of the n-th diffraction order of the light, with n an integer, and
• A second optical element having a structure for changing the phase of the 0th diffraction order of the light, wherein

at least the second optical element is connected to a drive circuit for changing the phase of the light during observation of the sample.

When the first optical element may be a rotatable spiral phase mask be provided only in the periphery of the spiral phase mask corresponds, as described above with reference to the prior art has been. The center of this spiral phase mask is recessed and according to the invention a reserved second optical element.

The second optical element is advantageous according to the invention as a nematic Liquid Crystal Display (LCD) formed with an electronic drive circuit connected is. In dependence of the control is the refractive index of the liquid crystals of this liquid Crystal displays changed and thereby the speed of propagation of the light so affected that the phase the 0th diffraction order by a predetermined phase angle within a phase angle range φ> 0 is delayed.

To For this purpose, the second optical element is in the center of the first positioned optical element.

With Such arranged in the imaging beam path optical Arrangement according to the invention is advantageously achieved, that too the phase of the 0th diffraction order is controllably changed and the spiral phase contrast method with higher Efficiency executed can be.

alternative This may, in a particularly preferred embodiment of the invention as the first optical element also at least one nematic Liquid Crystal Display (LCD) provided and also with a Be connected control circuit. This is dependent on from driving the alignment of the liquid crystals also this liquid Crystal displays varies, thereby the refractive index of these liquid crystals changed and thus affects the propagation speed of the light, thereby the phase of the nth diffraction order with n of an integer around one predetermined phase angle within a phase angle range φ> 0 is delayed.

So is advantageously generated a spiral phase contrast, the a quantitative determination of the phase without a mechanically moved, enables rotation of the optical element.

There the refractive index changes with the orientation of the liquid Crystals the nematic LCD's only changes in one polarization direction of the light, should be in the microscope beam path a polarizer can be arranged. The polarizer is preferred classified in the illumination beam path, in the context of the present However, the invention is also its classification in the imaging beam path.

in the In view of a space-saving design, it is recommended to use the polarizer on an optically effective Oberflä surface of an already in the beam path to arrange existing lens.

The Spiral phase mask lies in or near a lens pupil. So can For example, be provided, the LCD's or the spiral phase mask on a optically effective surface to arrange a lens in the lens.

Farther preferred is the phase contrast microscope according to the invention formed as a wide-field microscope, and the receiving means comprises a Camera for image acquisition and means for image evaluation.

in the Frame of the invention are also embodiments in which the Control circuit for the liquid Crystals the LCD's so formed is that the Control the liquid crystals in dependence of the wavelength the light coming from the light source takes place. This can both Lasers, LCD's or also white light sources as light sources for the Lighting light can be provided.

The Invention will be explained in more detail with reference to embodiments. In the associated Drawings show:

1 a first embodiment of the arrangement according to the invention for changing the phase of the light with a circular optical element arranged to the optical axis of a microscope objective circular optical element for controllably changing the phase of the 0th diffraction order and a concentric thereto rotatably arranged spiral phase mask having a structure for changing the phase of n -th order of diffraction,

2 a second embodiment, in the deviating from the embodiment according to 1 instead of the rotatable spiral phase mask, there is an optical element for controllably changing the phase of the n-th diffraction orders,

3 an example of the arrangement of the inventive arrangement according to 1 or after 2 in a microscope lens,

4 an example of the use of a microscope objective after 3 in a wide field microscope for the transmitted light method,

5 an example of the use of a microscope objective after 3 in a wide field microscope for the incident light method.

In 1 the arrangement according to the invention for changing the phase of the light in a first embodiment is shown. The arrangement here has a spiral phase mask arranged rotatably about the optical axis of an imaging beam path 1 on, which serves to influence the phase of the light passing through.

As already stated above, the spiral phase mask is 1 an optical element ^having a fixed phase structure that propagates in the circumferential direction U according to the function Φ (r, φ) = e ^{-1 φ} spiral. The through this spiral phase mask 1 Consequently, a phase delay is imparted regardless of the distance to the optical axis, wherein in each case the n-th and the -n-th diffraction order centrally symmetrical through the spiral phase mask 1 pass through and thereby be delayed by 180 ° to each other, but except the 0th order of diffraction.

The 0th diffraction order passes through the center and becomes independent of the rotation of the spiral phase mask 1 delayed by a constant amount in the phase about the optical axis. This delay of the phase by a constant amount has the disadvantage that the contrast of edge structures is weakened.

In this regard, the invention provides the spiral phase mask 1 to provide in their center with a circular recess, in this recess, for example, as a nematic liquid crystal display (LCD) executed optical element 2 to position and this with a drive circuit 3 which changes the refractive index of the liquid crystals depending on the size of a drive voltage applied to the liquid crystals, thereby affecting the propagation speed of the light and thus the phase of the 0th diffraction order. The influencing takes place within a phase angle range of at least φ to φ + 2π.

With such in the imaging beam path of a phase contrast microscope arranged optical arrangement is advantageously achieved that the spiral phase contrast method with higher efficiency accomplished can now be controlled because now also the phase of the 0th diffraction order and thus specifically changeable is.

2 shows a second embodiment of the inventive arrangement. Here is the center of the spiral phase mask 1 again, for example, as a nematic liquid crystal display (LCD) performed optical element 2 positioned and with a drive circuit 3 connected, which can influence the phase of the 0th diffraction order within a phase angle range of at least φ to φ + 2π by means of a drive voltage applied to the liquid crystals.

For the sake of clarity, in 2 For assemblies that basically serve the same purpose as the assemblies in 1 , also the same reference numerals as in 1 used. This also applies to the other illustrations in 3 to 5 ,

Unlike the spiral phase mask 1 according to the embodiment in 1 is the spiral phase mask 1 in 2 not provided with a fixed predetermined, in the circumferential direction U spirally propagating phase structure, but it consists of individual, in the circumferential direction U juxtaposed circle-shaped nematic LCD elements.

Both these nematic LCD elements and the nematic LCD element positioned in the center are provided with a driving circuit 3 connected, which is designed so that each of the nematic LCD elements can be separately and selectively controlled.

The phase structure of the spiral phase mask 1 to 1 thus changes stepwise depending on the driving and on the size in which the circular cutout nematic LCD elements are implemented.

In this case, the control of the peripherally arranged circular sector-shaped nematic LCD elements is always carried out so that the phase of the n-th diffraction order and the n-th diffraction order (with n an integer) as in the spiral phase mask 1 to 1 be delayed by 180 ° against each other.

This is compared to the embodiment according to 1 In addition, the advantage that the spiral phase mask 1 does not have to rotate about its center, but the rotation and thus the rotation of the spiral structure is simulated with the control of the peripheral LCD elements. So there are no mechanically moving and thus subject to wear modules required.

The control of the arranged in the center LCD element 2 takes place as well as in the embodiment variant 1 such that the 0th diffraction order is delayed in a phase angle range of at least φ to φ + 2π.

3 shows an example of the arrangement of an inventive arrangement according to the first or second embodiment in the pupil plane 4 a microscope objective 5 , centric to its optical axis 14 , Is symbolic Again, the connection of the arrangement according to the invention with a drive circuit 3 shown.

4 shows an example of the use of a equipped with the inventive arrangement microscope objective after 3 in transmitted light.

Indicated here are the spiral phase mask 1 (executed either after the in 1 or 2 described embodiment variant), in the center of the spiral phase mask 1 arranged nematic LCD element 2 , the drive circuit 3 and the pupil plane 4 of the microscope objective.

Furthermore, a light source can be seen 6 , a polarizer 7 , a pinhole 8th , a sample to be examined 9 , a camera serving as a receiving device 10 to which an imaging beam path 12 meets, as well as a calculator 11 equipped with a software for image analysis and both with the camera 10 as well as with the drive circuit 3 communicates.

Deviating from the illustration according to 4 is in 5 an example of the use of a equipped with the inventive arrangement microscope objective after 3 shown in the incident light method. Here are essentially the same functional assemblies as in 4 to recognize. However, the functional modules are associated with each other according to the incident light method. In addition, a beam splitter 13 provided for the decoupling of an imaging beam path 12 serves.

1

Spiral phase mask

2

optical Element / LCD element

3

drive circuit

4

pupil plane

5

microscope objective

6

light source

7

polarizer

8th

pinhole

9

sample

10

camera

11

computer

12

Imaging beam path

13

beamsplitter

14

optical axis

U

circumferentially

Claims (13)

Phase contrast microscope, comprising - a light source ( 6 ), - a sample to be observed, - optical means for generating an illumination beam path directed onto the sample from that of the light source ( 6 ), - optical means for generating an imaging beam path ( 12 ) from the light transmitted, reflected or scattered by the sample, - a receiving device to which the imaging beam path is directed, and - an optical arrangement arranged in the imaging beam path for changing the phase of the light, characterized in that - the arrangement for changing the Phase of the light - a first optical element having a structure for changing the phase of the n-th diffraction order of the light, with n an integer, and - a second optical element ( 2 ) having a structure for changing the phase of the 0th diffraction order of the light, wherein - at least the second optical element having a drive circuit ( 3 ) is connected to change the phase of the light during observation of the sample. Phase-contrast microscope according to Claim 1, in which as a second optical element ( 2 ) a nematic liquid crystal display (LCD) is provided and this with a drive circuit ( 3 ), depending on the control of the refractive index of the liq sigkristalle and thus the propagation speed of the light affected and so the phase of the 0th diffraction order within a phase angle range of at least φ to φ + 2π is delayed. Phase-contrast microscope according to Claim 1 or 2, in which the second optical element ( 2 ) is positioned in the center of the first optical element. Phase-contrast microscope according to Claim 3, in which the first optical element is a spiral-phase mask which can be rotated about the optical axis ( 1 ) is provided. Phase-contrast microscope according to Claim 3, in which at least one further nematic liquid crystal display (LCD) is provided as the first optical element, and this with the drive circuit ( 3 ), wherein, depending on the driving, the refractive index of the liquid crystals, thereby affecting the propagation speed of the light and thus delaying the phase of the n-th diffraction order, is denoted by n of an integer. Phase contrast microscope according to one of the aforementioned Claims, in which a polarizer, preferably in the illumination beam path, is available. Phase-contrast microscope according to claim 6, wherein the polarizer is disposed on an optically effective lens surface is. Phase-contrast microscope according to one of the preceding claims, in which the liquid crystal displays (LCDs) and / or the spiral phase mask ( 1 ) are arranged in or near an objective pupil. Phase-contrast microscope according to Claim 8, in which the liquid crystal displays (LCDs) and / or the spiral phase mask ( 1 ) are arranged on an optically effective lens surface near the objective pupil. Phase contrast microscope according to one of the aforementioned Claims, formed as a wide-field microscope. Phase-contrast microscope according to one of the preceding claims, in which the receiving device is a camera ( 10 ) for image acquisition and means for image analysis. Phase-contrast microscope according to one of the preceding claims, in which the light source ( 6 ) in each case at least one laser, an LED or a white light source is provided. Phase-contrast microscope according to one of the preceding claims, equipped with a liquid crystal display (LCD) liquid crystal drive circuit, which is designed such that the liquid crystals are dependent on the wavelength of the light source ( 6 ) coming light.