Optical microscope and method for providing structured illumination light
阅读说明:本技术 光学显微镜及提供结构化照明光的方法 (Optical microscope and method for providing structured illumination light ) 是由 格哈德·克拉姆伯特 凯·威克 拉尔夫·内茨 罗纳德·德克尔 埃德温·扬·克莱因 道维·哈门 于 2018-04-24 设计创作,主要内容包括:一种光学显微镜,包括:结构化光学单元,包括用于提供结构化照明的波导芯片;输入选择设备,用于将光可变地引导到波导芯片的多个输入端的其中之一;波导芯片还包括跟随每个输入端的光导路径;每条光导路径分为几个路径分支;且每一路径分支引向波导芯片的一个输出端。波导芯片的输出端可设置在光学显微镜的光瞳平面处,且来自输出端的光的出射方向垂直于由波导芯片定义的平面。还描述了一种使用光学显微镜提供结构化照明光的方法。(An optical microscope, comprising: a structured optical unit comprising a waveguide chip for providing structured illumination; an input selection device for variably directing light to one of a plurality of input ends of the waveguide chip; the waveguide chip further comprises a light guiding path following each input end; each light-guiding path is divided into several path branches; and each path branch leads to one output of the waveguide chip. The output end of the waveguide chip may be disposed at a pupil plane of the optical microscope, and an exit direction of light from the output end is perpendicular to a plane defined by the waveguide chip. A method of providing structured illumination light using an optical microscope is also described.)
1. An optical microscope, comprising:
-a structured optical unit for providing structured illumination from incident light, the structured optical unit comprising a waveguide chip (90) having a plurality of input ends (31, 41, 51);
-an input selection device (8) for variably directing light to one of the input ends (31, 41, 51);
-a waveguide chip (90), further comprising a light-guiding path (32, 42, 52) following each input end (31, 41, 51), wherein each light-guiding path (32, 42, 52) is divided into a plurality of path branches (34, 35, 36; 44, 45, 46; 54, 55, 56) and each path branch (34, 35, 36; 44, 45, 46; 54, 55, 56) leads to an output end (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip;
it is characterized in that the preparation method is characterized in that,
the exit direction (75) of the light from the output ends (37, 38, 39, 47, 48, 49, 57, 58, 59) is perpendicular to a plane (P) defined by the waveguide chip (90).
2. The optical microscope of claim 1,
the light-conducting paths (32, 42, 52) and the path branches (34, 35, 36; 44, 45, 46; 54, 55, 56) extend in or parallel to a pupil plane of the optical microscope.
3. The optical microscope of claim 1 or 2,
each output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) comprises an interface (71) for deflecting light out of the waveguide chip (90) by total internal reflection.
4. The optical microscope of claim 1 or 2,
each output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) comprises an interface (71) for deflecting light out of the waveguide chip (90), wherein the interface (71) comprises a mirror.
5. The optical microscope of claim 3 or 4,
each interface (71) is angled between 20 ° and 70 ° with respect to the plane (P) of the waveguide chip (90).
6. The optical microscope according to any one of claims 3 to 5,
the interface (71) is formed by a recess on the waveguide chip (90).
7. The optical microscope according to any one of claims 1 to 6,
the path branches (34, 35, 36; 44, 45, 46; 54, 55, 56) are formed such that at least some (36, 46) of the path branches (34, 35, 36; 44, 45, 46; 54, 55, 56) belonging to different input ends (31, 41) intersect; and is
The intersecting path branches (36, 46) intersect at an angle between 70 ° and 120 °.
8. The optical microscope according to any one of claims 1 to 7,
the output ends (37, 38, 39) of the waveguide chips (90) belonging to the same input end (31) form a dot pattern, and the dot patterns are similar to each other but rotated with respect to each other.
9. The optical microscope of any one of claims 1 to 8,
further comprises an input polarizing unit configured to polarize the light such that, when incident on the input end (31, 41, 51) of the waveguide chip (90), its polarization direction is in the plane (P) of the waveguide chip (90).
10. The optical microscope of any one of claims 1 to 9,
further comprising an output polarization unit (25) on which light from the output ends (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) is incident,
the output polarization unit (25) is configured to rotate the polarization direction of the incident light by 90 °, in particular the output polarization unit (25) comprises two half-wave plates arranged in series and rotated by 45 ° with respect to each other.
11. The optical microscope of any one of claims 1 to 10,
each output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) comprises two mirrors arranged at a substrate (70) of the waveguide chip (90),
the mirror is configured to rotate the polarization direction of the incident light by 90 °.
12. The optical microscope of any one of claims 1 to 11,
the waveguide chip (90) comprises, on some or all of the path branches (34, 35, 36, 44, 45, 46, 54, 55, 56), a tunable phase shifter, in particular a piezoelectric or pyroelectric phase shifter, for adjustably setting a phase shift of the light in the respective path branch (34, 35, 36; 44, 45, 46; 54, 55, 56).
13. The optical microscope of any one of claims 1 to 12,
to provide TIR illumination, the waveguide chip (90) comprises one or more additional input ends (61, 62, 63), each connected to an additional light guiding path (64, 65, 66) leading to a respective TIR output end (67, 68, 69) of the waveguide chip (90),
wherein the output ends (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) define a geometric center, and
wherein each TIR output (67, 68, 69) is further from the geometric center than any output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90).
14. The optical microscope of any one of claims 1-13, further comprising:
a zoom assembly (18) disposed behind the output end (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90),
a control unit designed to receive a control command indicating whether structured illumination or total internal reflection illumination is required,
if total internal reflection illumination is required, the control unit is designed to set a greater magnification using the zoom assembly (18).
15. The optical microscope of any one of claims 1-14, further comprising:
-an optical element (19) arranged between the waveguide chip (90) and the sample plane (20) and configured to:
-creating a pupil plane at the position of the waveguide chip (90),
-wherein the output ends (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) are arranged in particular at a pupil plane, and
-generating, in the sample plane (20), an interference pattern of light exiting through the output ends (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) to provide structured illumination (20) in the sample plane.
16. The optical microscope of any one of claims 1 to 15,
each light-guiding path (32, 42, 52) is connected to a splitter (33, 43, 53) formed in the waveguide chip (90) and configured to divide the light-guiding path (32, 42, 52) into four parts,
wherein three of the portions constitute three path branches (34, 35, 36; 44, 45, 46; 54, 55, 56) and one of the portions (30, 40, 50) directs light away so that the portion of light does not illuminate the sample.
17. A method of providing structured illumination light within an optical microscope, the method comprising the steps of:
-directing light (5) from a light source (4) to an input selection device (8);
-variably directing light with an input selection device (8) to one of a plurality of input ends (31, 41, 51) of a waveguide chip (90) configured to provide structured illumination from incident light (5);
-the waveguide chip (90) further comprises a light-guiding path (32, 42, 52) following each input end (31, 41, 51), wherein each light-guiding path (32, 42, 52) is divided into a plurality of path branches (34, 35, 36; 44, 45, 46; 54, 55, 56); and each path branch (34, 35, 36; 44, 45, 46; 54, 55, 56) leading to one output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90);
it is characterized in that the preparation method is characterized in that,
-the light leaves the output end (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) in an exit direction (75) perpendicular to the plane (P) defined by the waveguide.
18. The method of claim 17,
the output (37, 38, 39, 47, 48, 49, 57, 58, 59) of the waveguide chip (90) is arranged at or in the region of a pupil plane of the optical microscope.
Technical Field
The disclosure of the present invention relates to an optical microscope having the features of the preamble of claim 1. The optical microscope is configured to convert light from a light source into structured illumination.
The disclosure also relates to a method for providing structured illumination light having the features of the preamble of
Background
Structured Illumination Microscopy (SIM) is an established technique for examining samples with exceptionally high resolution. Structured lighting is generally considered to be an arbitrary light distribution that is not uniform across the cross-section. For example, the structured illumination may include one or more lines, a raster pattern, or only points having a non-uniform intensity profile. These patterns are generated on the sample plane.
If a plurality of images using different structured illumination patterns are recorded, an image of enhanced resolution can be calculated from these images. Typically, nine or more images (referred to as raw images or a frame) are recorded and a high resolution image is calculated therefrom. These original images differ in the direction of rotation of the structured illumination and in the phase shift of the structured illumination. The enhanced resolution may be above the diffraction limit or abbe limit for the wavelengths used. It is often desirable that the different structured illumination patterns be similar but rotated relative to each other.
In the context of this document, structured illumination is not limited to a given SIM technology, but may also be used in related technologies, such as synthetic aperture imaging, which may examine a sample using different patterns of structured illumination.
In many conventional methods, the structured elements are arranged in an intermediate image plane (mtp). For example, the structured elements may be gratings. The grating pattern is thus imaged into the sample plane. To record the different images, the structuring element may be rotated. Optionally, an image rotator may be introduced into the optical path and/or the image rotator is rotating. It is also possible to produce patterns in different directions in the sample plane by using several different structured elements, which are optionally introduced into the light path. These methods involve rotation or other movement of rather large and heavy parts, such as structured elements or image rotators. Such motion constitutes a serious speed limitation. An advantageous solution to reduce the time requirement in recording a plurality of images is described by the applicant's patents DE 102007047466 a1 and DE 102012020877 a 1. Each of these applications describes an optical assembly in which structured illumination is not produced by arranging structured elements in a primary image plane. Instead, a spot light pattern or a light spot pattern is generated on the focal plane. The pattern in the focal plane results in a different structured pattern in the sample plane by means of a fourier transformation. Thus, the spot light pattern may result in a stripe or grid pattern, as produced with a grating in a primary image plane.
In DE 102007047466 a1, different spot patterns are produced using optical fibers that introduce light into the pupil plane through microlenses or other optical elements. Depending on which fibers are illuminated, different spot patterns can be produced in the pupil plane.
As described in patent DE 102012020877 a1, a common optical microscope comprises:
-a structured optical unit for providing structured illumination from incident light, the structured optical unit comprising a waveguide chip having a plurality of input ends;
-an input selection device for variably directing light to one of the input ends;
the waveguide chip further comprises a light guiding path following each input end; each light-guiding path is divided into a plurality of path branches; and each path branch leads to one of the input ends of the waveguide chip.
A common method for providing structured light illumination in an optical microscope comprises the steps of:
-directing light of a light source to an input selection device;
-using an input selection device to variably direct light towards one of a plurality of input ends of a waveguide chip configured to provide structured illumination from incident light;
the waveguide chip further comprises a light guiding path following each input end; each light-guiding path is divided into a plurality of path branches; and each path branch leads to one of the input ends of the waveguide chip.
Thus, ordinary optical microscopes do not use gratings or other structuring elements as structured optical units in the primary image plane. The waveguide chip is used instead: such a chip comprises a substrate, for example fused silica, in which a light-guiding path or path branch is formed. The material of these paths has a different refractive index than the surrounding substrate. This may be achieved by doping the substrate in the areas where the paths are to be formed.
With an input selection device, different inputs can be illuminated in sequence. By dividing each light guiding path into a plurality of path branches, coherent light can be transmitted simultaneously through a plurality of output ends (belonging to the same input end). Distributing the light from the path into the path branches will not require any (movable) switching elements within or on the waveguide chip. Thus, a path branch may be defined as one of a plurality of paths in the waveguide chip connected to one light guiding path. The light propagating in the light-guiding path will be spread into the plurality of paths, which are thus called path branches.
Patent DE 102012020877 a1 uses an optical fiber connected to the output end to direct the light towards the pupil plane. The ends of these fibers are arranged in a desired spot pattern in the pupil plane. The spot pattern is converted into the desired structured illumination pattern on the image plane.
However, known techniques for generating a spot pattern in a pupil plane require a demanding and costly setup: the use of optical fibers with ends arranged in the pupil plane places high demands on the positioning accuracy, which long-term stability may become a problem. In addition, the variation in fiber length must be minimal due to the coherence length of the laser.
It can therefore be seen as an object of the present invention to provide an optical microscope and a method for providing structured illumination light which provide particularly good stability while also reducing the complexity of the setup.
Disclosure of Invention
The above object is solved by an optical microscope comprising the features of claim 1 and a method comprising the features of
According to the present invention, the above-described general optical microscope is characterized in that the exit direction of light from the output end is perpendicular to the plane defined by the waveguide chip. This allows the output to be formed at the exact position required for a certain spot pattern in or near the pupil plane. Therefore, it is not necessary to rearrange the light pattern at the exit output: it is suggested in DE 102012020877 a1 to use optical fibers to follow the output end to guide the light to the pupil plane and to rearrange the light pattern, i.e. the end of the optical fibers (not facing the waveguide chip) defines the light pattern in the pupil plane. The present invention makes the use of such optical fibers obsolete, thus avoiding all the problems associated with proper alignment, accuracy and long-term stability of such fiber optic devices. In contrast, the invention allows the waveguide chip to be arranged with its output end in the pupil plane of the optical microscope. One key feature is the lateral coupling of light into the plane defined by the waveguide chip. It can be considered that this plane is spanned by two longer sides of the waveguide chip, which can be referred to as length and width, which means that light is coupled out in the height direction of the waveguide chip. Alternatively, a plane may be defined as being traversed by the directions of the light-guiding paths and the path branches. Preferably, this plane is parallel to the pupil plane.
In other words, the direction of the coupled-out ends of the light is perpendicular to the direction of the respective light-guiding paths/path branches, i.e. perpendicular to the plane spanned by these paths. In contrast to the prior art, where the light beam leaves the waveguide chip at one of its smaller sides, the present invention provides a waveguide chip, where the light exits at the largest side, i.e. the plane defined above. The coupling-out or exit direction is perpendicular to this plane, which includes any tilt angle, and may especially be perpendicular, in which case the plane defined by the waveguide chip may be exactly in the pupil plane and parallel thereto. The light-conducting path and the path branches can also extend in the pupil plane of the optical microscope or be parallel to the pupil plane. The tilt angle may be considered to consist of any angle between 0 ° and 80 ° relative to the plane normal or any angle between 0 ° and 70 ° relative to the plane normal.
A common method as described above is characterized in that the light exits the output end in an exit direction transverse to the plane defined by the waveguide chip. Preferably, the output end of the waveguide chip can be arranged in the pupil plane or in a region in the pupil plane of the optical microscope. The arrangement of the waveguide chip in the region of the pupil plane is understood to mean that the pupil plane intersects the plane of the waveguide chip or, alternatively, the distance from the pupil plane to the light guide plate is less than the maximum distance at which the light beam exits the light guide plate through the output of the waveguide chip.
Drawings
Further features and characteristics of the present invention are described with reference to the accompanying drawings.
Fig. 1 schematically shows an embodiment of an optical microscope with an optical assembly according to the invention;
fig. 2 schematically shows an embodiment of the optical assembly of fig. 1. (ii) a
FIG. 3 schematically illustrates an exemplary design of a portion of the optical assembly of FIG. 1 or FIG. 2;
fig. 4 schematically shows another embodiment of an optical assembly of an optical microscope according to the invention;
FIG. 5 schematically illustrates the position and polarization direction of a light beam at or behind a waveguide chip of an optical microscope of the present invention;
fig. 6 schematically illustrates another exemplary design of a portion of the optical assembly of fig. 1 or 2.
Throughout the drawings, similar components have the same reference numerals.
Detailed Description
Fig. 1 schematically shows an embodiment of an optical microscope 1 of the invention, comprising an
Fig. 1 schematically shows an embodiment of an optical microscope 1 of the invention, comprising an
The microscope 1 comprises a
In the depicted example, the input selection device 8 is formed as a scanner 8 with a rotatable mirror. However, in the following description, the scanner 8 may also be replaced by other variations of the input selection device described elsewhere in the present disclosure.
As shown in fig. 1, the
The scanner 8 is configured to variably deflect the
The
The design of the
As an important feature of the present invention, the output of the
The structured
As shown in fig. 1, the microscope 1 may further include a
The
Light from the sample is detected by a detector or
A
The microscope 1 may further comprise a pyroelectric or piezoelectric phase shifter, preferably integrated in the
Turning to fig. 2, the design of an
Each light-guiding
In the depicted example, there are first, second and
The
The pattern of dots in the pupil plane spatially corresponds to the light beams of different diffraction orders of the grating arranged in the primary image plane. In prior art arrangements, such gratings are used to provide structured illumination. The light diffracted on the grating forms a plurality of beam portions corresponding to different diffraction orders. The diffraction orders comprise in particular the zeroth diffraction order as part of the central light beam and the negative first and first diffraction orders. In the pupil plane, these zero, negative first and first diffraction orders may form three points along a line. The output terminals 37-39, 47-49, 57-59 are arranged to produce such a dot pattern in the pupil plane. For easier understanding, reference will be made below only to the outputs 37-39. The
The light portions at these outputs 37-39 interfere with the sample plane. The relative light intensities at the output ends 37-39 are important in order to have particularly good contrast in such interference patterns. The
The above description of outputs 37-39 and their connected components also applies to outputs 47-49 and 57-59.
The arrangement of the dot patterns of the groups of these output terminals 37-39, 47-49 and 57-59 differs from one another. Each dot pattern may be formed of dots on one line; however, the different sets of wires of the outputs 37-39, 47-49 and 57-59 are rotated relative to each other. In other words, each
The different point patterns rotated with respect to each other correspond to structured patterns in the sample plane rotated with respect to each other, which is required for structured illumination microscopy.
A key feature of the
This will be further explained with reference to fig. 3, which schematically shows details of the
Fig. 3 shows a
Surface 71 may also be referred to as a TIR (total internal reflection) micromirror. If the surface 71 is the interface between air and the substrate/path branch, the evanescent optical field penetrates the air. The evanescent field may be, for example, about 100 nanometers for commonly used wavelengths of light. The evanescent field may cause degradation of the surface 71 due to interaction with air molecules. To avoid such defects, surface 71 may be coated (e.g., with a metal or dichroic coating) to avoid interaction of the evanescent field with air. Optionally, the recess 72 may be provided with a cover and filled with a protective gas such as argon. While surface 71 is in contact with a shielding gas that does not interact with the evanescent field.
In addition to total internal reflection, mirrors may be used at the interface 71.
In other variations of the invention, the surface/interface 71 may be formed at another angle to cause an exit direction that varies from 90 °.
The reflective interface 71 is formed on one side of the substrate such that the reflected light passes through the substrate before exiting the
The interface or mirror 71 is arranged such that the reflected light is directed through the
Other path branches and other outputs may be formed similarly to that shown in fig. 3.
Although fig. 2 shows an embodiment in which each
Fig. 4 shows another embodiment of a
The features of the splitter design and the input for TIR illumination are independent of each other, and therefore the optical assembly of fig. 2 may be additionally provided with the splitter design of fig. 4 and/or the features for TIR illumination as explained with reference to fig. 4.
The
As depicted in fig. 2, only three of the four path branches 34-36 lead to the output. Each remaining
The remaining
Optionally, the
In further embodiments, the
Other remaining
Instead of four path branches, another even number may be used, wherein in each case one path branch is not used for illuminating the sample.
If one of the
Furthermore, the
The
A respective optical fiber may be directed to each TIR input 61-63 so that scanner 8 can select any of
Although fig. 4 shows an embodiment with three TIR inputs 61-63, the design may be generalized to one or more TIR inputs.
The efficiency of each splitter depends on the polarization of the incident light. Preferably, the light is linearly polarized in the direction in which the separation occurs (as indicated by the arrows in fig. 2 and 4). However, the SIM requires a polarization that is preferably perpendicular to this direction. Therefore, it is preferable to rotate the polarization of the light after the beam splitter.
Further described with reference to fig. 5, three dot patterns are shown in the left hand portion. The first spot pattern comprises
The polarization direction of each spot 81-89 is indicated by an arrow.
Two half-wave plates can be used, in each case with a rotation of the polarization direction by 90 °. As shown in fig. 1, such a half-
One or both of the half-wave plates may also be replaced with other ways of achieving a 90 ° polarization rotation. Turning to fig. 3, the substrate surface at each
Each path branch may also lead to two consecutive mirror interfaces before the light exits the waveguide chip. An example is shown in fig. 6, which schematically shows a perspective view of a portion of a
The
In addition to the described light-conducting path, a further light-conducting path is provided which is not connected to one of the input ends 31, 41, 51 of the waveguide chip. The further light guiding path may have an input coupled to a further light source, for example an LED connected to a waveguide chip at the input of the further light guiding path. The other light-guiding path has an output end located near one of the input ends 31, 41, 51. For example, the distance between the output end of the further light-guiding path and one of the input ends 31, 41, 51 may be smaller than the diameter of the five (more) light-guiding paths. In particular, the out-coupling direction of the output end of the further light-guiding path may be parallel to the in-coupling direction of the input ends 31, 41, 51. This may be done by another light-guiding path at the input end of its cross-section, parallel to the cross-section of the light-guiding
The design of the present invention provides improved stability and accuracy because the output end of the waveguide chip and the pattern of dots in the pupil plane have a predetermined setting and no optics are required to direct light from the waveguide chip to the pupil plane, which would require alignment or may be subject to misalignment over time. In particular, no optical fiber is required for this purpose, thus avoiding coherence problems due to different lengths of optical fiber. As a result, a less expensive laser can be used as the light source. This can be achieved using an arrangement requiring a relatively low number of components.
The main advantage of the optical microscope of the present invention is that many functions can be integrated in a single waveguide chip. This not only allows a reduction in space but also avoids adjustment problems of the individual movable parts. For example, the optical fiber behind the waveguide chip becomes obsolete. The phase shifter and/or polarization rotation means may also be integrated in the waveguide chip; wherein the polarization rotation means may be formed by a micromirror shape or by a mirror image pair of half-wave plates provided at each output end.
List of reference numerals
1 optical microscope
4 light source
5 light
6 mirror for combining beam paths
7AOTF
8 scanner
9. 16 reflective surface of movable deflector
10 dichroic beam splitter
11.1, 11.2, 11.3 optical fiber
15 structured light exiting a waveguide chip
18 zoom assembly
19 objective lens
20 sample plane
21 control unit
22 Detector
23 lens
24-dichroic beam splitter
25 output polarization cell with half-wave plate
26 optical filter
27 Movable deflector
28 Camera
30. 40, 50 path branches not used for illuminating the sample
31. Input end of 41, 51 waveguide chip
32. 42, 52 light guide path
33. 43, 53 optical splitter formed in waveguide chip
34. 35, 36; 44, 45, 46; 54. path branching for 55, 56 waveguide chips
37. 38, 39, 47, 48, 49; 57. 58, 59 waveguide chip output
61. 62, 63 additional inputs
64. 65, 66 additional light guide paths
67. 68, 69TIR output
70 waveguide chip substrate
71 interface for deflecting light out of a waveguide chip
72 recess provided in the substrate of the waveguide chip
73 interface for deflection within a waveguide chip
75 outgoing direction of light at output end
Polarization direction of light in 76-waveguide chip
77 polarization direction of light coupled out of the waveguide chip
Spots in pupil planes 81-89
90 waveguide chip
95 optical assembly
96. Fast axis of 97 half-wave plate
Plane defined by P-waveguide chip