Light Sheet Microscope and Method for Operating Same

Abstract

A microscope including an illumination objective with a first optical axis, embodied to produce a light sheet, and a detection objective with a second optical axis, embodied to detect light coming from the specimen plane. The illumination objective and the detection objective are aligned relative to one another and the specimen plane so that the first and second optical axes intersect in the specimen plane and include a substantially right angle therebetween. The optical axes each include an angle which differs from zero with a reference axis directed orthogonal to the specimen plane. An overview illumination apparatus for wide-field illumination of the specimen plane, includes an illumination optical unit with a third optical axis. The characterizing feature is that the detection objective is provided to detect both light from the light sheet and light from the illumination optical unit. A method is also provided for operating a light sheet microscope.

Claims

1. A microscope comprising: an illumination objective, with a first optical axis, configured to produce a light sheet at least in part in a specimen plane; a detection objective, with a second optical axis, configured to detect light coming from the specimen plane; and an illumination optical unit for wide-field illumination of the specimen plane, the illumination optical unit comprising an illumination optical unit with a third optical axis; wherein the illumination objective and the detection objective are aligned relative to one another and relative to the specimen plane in such a way that the first optical axis and the second optical axis intersect in the specimen plane (4) and form a substantially right angle therebetween; wherein the first optical axis and the second optical axis (A2) each form an angle which differs from zero with a reference axis orthogonal to the specimen plane; and wherein the detection objective is configured to detect both light from the light sheet, and light from the illumination optical unit.

2. The microscope according to claim 1; wherein the third optical axis of the illumination optical unit is directed substantially along the reference axis.

3. The microscope according to claim 1, further comprising a mask that is arranged in a pupil of the illumination optical unit.

4. The microscope according to claim 1, further comprising: a polarizer that is arranged in a beam path of the illumination optical unit and in a beam path of the detection objective.

5. The microscope according to claim 1, further comprising: Hoffmann optical units that are arranged in a beam path of the illumination optical unit and in a beam path of the detection objective.

6. The microscope according to claim 1, further comprising: DIC prisms that are arranged in a beam path of the illumination optical unit and in a beam path of the detection objective.

7. The microscope according to claim 1, further comprising: a diffusor that is arranged between the detection objective and the illumination optical unit.

8. The microscope according to claim 7; wherein the diffuser comprises an LED, or an OLED with a ground glass screen disposed downstream thereof.

9. The microscope according to claim 1, further comprising: a mask arranged in a beam path of the illumination optical unit, said mask configured to darken an overlap region so as to produce dark-field illumination in the specimen plane.

10. The microscope according to claim 1, further comprising: a movable mask arranged in a pupil of the illumination optical unit, exactly one half of the pupil being covered by said mask.

11. A method for operating a light sheet microscope, comprising the following steps: illuminating a specimen situated in a specimen plane with light from an overview illumination apparatus along a reference axis directed substantially orthogonal to the specimen plane; detecting the light from the overview illumination apparatus as transmitted light by means of a detection objective having an optical axis, wherein the optical axis of the detection objective includes an angle that differs from zero with the reference axis; creating an overview image of the specimen depending on the light from the overview illumination apparatus, captured by means of the detection objective; and capturing light of a light sheet produced in a specimen plane by means of the detection objective to capture the light of the overview illumination apparatus.

12. The method according to claim 11, wherein the overview image is created by means of a TIE (transport of intensity equation).

13. The method according to claim 11, further comprising: capturing a Z-stack of XY-planes, wherein the XY-planes are transformed into a normalized Z-stack if they have a preferred direction, the XY-planes of said normalized Z-stack not having a preferred direction, by virtue of: the captured Z-stack being virtually surrounded by a lattice having X-, Y- and Z-axes that extend orthogonal to one another, wherein the X-axis and the Y-axis are directed parallel to the specimen plane and the Z-axis is directed perpendicular to the specimen plane; wherein the spacings of the XY-planes of the normalized Z-stack are selected in the direction of the Z-axis in such a way that the spacings correspond to one of the lateral resolutions of the captured Z-stack, such that the following applies:
Δx′=Δx;
Δy′=Δy; and
Δz′=Δx or Δy; calculating new lattice points (P.sub.x,y,z); and calculating an intensity at the respective new lattice points (P.sub.x′,y′,z′) by means of three weighted interpolations of adjacent (lattice) points (P.sub.x′,y′,z′) of the captured Z-stack.

14. The method according to claim 11, further comprising: capturing a Z-stack of XY-planes is captured, wherein the XY-planes are transformed into a normalized Z-stack if they have a preferred direction, the XY-planes of said normalized Z-stack not having a preferred direction, by virtue of: the captured Z-stack being virtually surrounded by a lattice having X-, Y- and Z-axes that extend orthogonal to one another, wherein the X-axis and the Y-axis are directed parallel to the specimen plane and the Z-axis is directed perpendicular to the specimen plane; wherein the spacings of the XY-planes of the normalized Z-stack are selected in the direction of the Z-axis (Z) in such a way that the spacings correspond to one of the lateral resolutions of the captured Z-stack, such that the following applies:
Δx′=Δx;
Δy′=Δy; and
Δz′=Δy*sin(α.sub.1); where the angle α.sub.1 is included by the first optical axis and the third optical axis; calculating new lattice points (P.sub.x,y,z); and calculating an intensity at the respective new lattice points (P.sub.x,y,z) by means of three weighted interpolations of adjacent (lattice) points (P.sub.x′,y′,z′) of the captured Z-stack.

15. The method according to claim 11; wherein a recording speed of the capture of an XY-plane is set by virtue of: (a) an increment Δz′ between two XY-planes to be captured being set; or (b) a Z-stack being captured with a first increment (Δz′), a region of interest being selected and the selected region of interest being captured with a second increment (Δz′), wherein the second increment (Δz′) is greater than the first increment (Δz′); or (c) only one XY-plane parallel to the specimen plane (4) being calculated and displayed in each case.

16. The method according to claim 11, further comprising: selecting a single-line region of interest in the direction of the X-axis or the Y-axis and respectively capturing the single-line region of interest per XY-plane in the direction of the Z-axis.

17. The method according to claim 15, alternative (c); wherein a respective XY-plane is depicted displaced by a value Δ=Δz′/tan(α.sub.1) in relation to the previous XY-plane.

17. The method according to claim 16; wherein the respective XY-plane is depicted displaced by a value Δ=Δz′/tan(α.sub.1) in relation to the previous XY-plane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1a shows a schematic illustration of a microscope with a 45° arrangement of the illumination objective and detection objective above a specimen plane and with a wide-field objective below a specimen plane, in accordance with the prior art;

[0072] FIG. 1b shows a schematic illustration of a microscope with an inverted 45° arrangement of the illumination objective and detection objective below a specimen plane and with a wide-field objective above a specimen plane, in accordance with the prior art;

[0073] FIG. 2a shows a schematic illustration of a first exemplary embodiment of a microscope according to the invention;

[0074] FIG. 2b shows a schematic illustration of the numerical apertures of the first exemplary embodiment of the microscope according to the invention;

[0075] FIG. 3a shows a schematic illustration of a second exemplary embodiment of a microscope according to the invention, comprising a diffusor;

[0076] FIG. 3b shows a schematic illustration of the numerical apertures of the second exemplary embodiment of the microscope according to the invention;

[0077] FIG. 4a shows a schematic illustration of a third exemplary embodiment of a microscope according to the invention, comprising a mask;

[0078] FIG. 4b shows a schematic illustration of the numerical apertures of the third exemplary embodiment of the microscope according to the invention;

[0079] FIG. 5a shows a schematic illustration of a fourth exemplary embodiment of the microscope according to the invention, comprising a half-sided mask;

[0080] FIG. 5b shows a schematic illustration of the fourth exemplary embodiment of the microscope according to the invention, in a side view and with schematically illustrated pupil coverages;

[0081] FIG. 6 shows a schematic illustration of a (fifth) exemplary embodiment of the microscope according to the invention, with symbolized capture regions;

[0082] FIG. 7a shows a schematic illustration of a Z-stack from a view along an optical axis of a detection objective of a microscope according to the invention;

[0083] FIG. 7b shows a schematic illustration of the Z-stack in a lateral view of the specimen;

[0084] FIG. 8a shows a schematic illustration of a displaced captured Z-stack (skew) and of a virtual lattice;

[0085] FIG. 8b shows a schematic illustration of a first transformation 1 and

[0086] FIG. 8c shows a schematic illustration of a second transformation 2.

[0087] The same reference signs denote the same elements in the following exemplary embodiments and schematic illustrations.

DETAILED DESCRIPTION OF EMBODIMENTS

[0088] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein.

[0089] The present invention will now be described in detail on the basis of exemplary embodiments.

[0090] An upright microscope 1, as depicted schematically in FIG. 1a, comprising an illumination objective 2, a detection objective 3 and a wide-field objective 20 is known from the prior art. A light sheet 6 is produced or producible along a first optical axis A1 by means of the illumination objective 2, said light sheet being usable to examine a specimen 5 arranged in a specimen plane 4. The detection objective 3 has a second optical axis A2, along which the light coming from the specimen plane 4 may be captured. The first optical axis A1 and the second optical axis A2 are aligned orthogonal to one another and each include an angle of 45° with the specimen plane 4 which serves as a reference plane B.

[0091] The wide-field objective 20 has a third optical axis A3, which is directed orthogonally to the specimen plane 4 and serves as a reference axis B. The first to third optical axes A1 to A3 intersect in the region of extent of the light sheet 6 in the specimen 5. Moreover, the first optical axis A1 includes a first angle α1 with the reference axis B and the second optical axis A2 includes a second angle α2 of 90°-α1 with the reference axis B, e.g. respectively 45°.

[0092] The specimen 5 is held in a specimen holder 7 which is filled with a liquid 8.

[0093] FIG. 1b schematically shows a microscope 1 with an inverted arrangement of illumination objective 2 and detection objective 3, in which the illumination objective 2 and the detection objective 3 are arranged below the specimen plane 4 and the wide-field objective 20 is arranged above the specimen plane 4. Once again, the angles α1 and α2 are 45° in each case.

[0094] The following exemplary embodiments are illustrated in an exemplary manner on the basis of inverted microscopes 1 and may, in further embodiments, also be embodied as upright microscopes 1.

[0095] In an inverted microscope 1 schematically depicted in FIG. 2a, the illumination objective 2 and the detection objective 3 are arranged at angles α1 and α2 of 45°.

[0096] In further embodiments of the microscope 1, the angles α1 and α2 have numbers of degrees deviating from 45°, with the angles substantially complementing one another to 90°.

[0097] Instead of a wide-field objective 20 (see FIGS. 1a and 1b), an illumination optical unit 9 of an overview illumination apparatus in the form of a condenser is present, said illumination optical unit being embodied to transmit light into the specimen plane 4 along the third optical axis A3, which coincides with the reference axis B. In further embodiments, the condenser is embodied as an air condenser. In further embodiments, the illumination optical unit 9 is embodied as an optical lens.

[0098] In further embodiments, the illumination optical unit 9 is formed by an illumination objective 20. In addition to illumination purposes, the latter may also be embodied for observing and/or imaging the specimen 5.

[0099] The illumination optical unit 9 is used to illuminate a region of the specimen 5 which lies in the field of view of the detection objective 3. Since the third optical axis A3 of the illumination optical unit 9 is aligned at an angle unequal to 0° or 180° in relation to the second optical axis A2, an oblique illumination is necessarily present without corresponding masks being introduced into the beam path of the illumination optical unit 9, as is usual in the case of conventional stands.

[0100] The oblique illumination facilitates capturing a wide-field image by means of the detection objective 3 as an overview image with an enhanced contrast. The illumination optical unit 9 need not necessarily be immersed into the liquid 8, e.g. water, of the specimen holder 7 (water dipping). Rather, the specimen 5 may be illuminated through the air/liquid interface using an (air) condenser. This is possible as the aberrations in the illumination, occurring in the process, are uncritical for imaging.

[0101] In further embodiments of the microscope 1, a polarizer, Hoffmann optical units and/or DIC prisms are optionally respectively arranged in suitable combinations in the beam path of the illumination optical unit 9 and in a beam path of the detection objective 3; this is shown schematically by the frame denoted by the reference sign 10.

[0102] FIG. 2b depicts the aperture cones, i.e. the numerical apertures NA1 or of the illumination optical unit 9 and NA2 of the detection objective 3, and a region in which the numerical apertures NA1, NA2 overlap (overlap region). The first optical axis A1 and the second optical axis A2 do not extend parallel to one another.

[0103] The numerical aperture NA1 of the illumination optical unit 9 is depicted schematically both as an object-side aperture angle and as an image-side aperture angle along the third optical axis A3.

[0104] This form of representing numerical apertures is also used in the following exemplary embodiments.

[0105] A diffuser 12 in the form of a diffusing screen is arranged in the beam path of the illumination optical unit 9 between the latter and the specimen 5 (FIG. 3a). The light of the illumination optical unit 9 scattered by the diffuser 12 is elucidated by half an oval edged by dashed lines (FIG. 3b).

[0106] Only a portion of the illumination light from the region of the overlap is detectable by means of the detection objective 3, depending on the numerical apertures NA1, NA2 and the mutual overlap thereof. It is possible to set the degree of overlap by adapting the numerical aperture NA1 of the illumination objective 20 or of the illumination optical unit 9. In order to achieve a resolution which is as high as possible, it is necessary to work with high numerical apertures NA1 and NA2, both on the illumination side and on the detection side. However, the strong overlap of NA1 and NA2 may be disadvantageous in the case of specimens with a low structure density, for example those with singulated, small objects, since it is necessary to detect small variations in front of a very bright background.

[0107] This impairment is avoided by virtue of a mask 11 being arranged in the pupil of the overview illumination apparatus or of the illumination optical unit 9, said mask precisely blocking the overlap region of NA1 and NA2, as depicted schematically in FIG. 4a.

[0108] In FIG. 4b, the region of the numerical aperture NA1 masked by means of the mask 11 is denoted by NA1mask and the unmasked region is denoted by NA1unmask.

[0109] Therefore, the mask may block the overlap region as plotted; in that case, a dark field is realized, in which only light scattered in the specimen 5 is detected by the detection objective 3. Alternatively, the non-overlapping region may be blocked by means of the mask 11, as a result of which an ideal oblique illumination is then obtained.

[0110] In a fourth exemplary embodiment of the microscope 1 depicted schematically in FIG. 5a, a mask 11 is arranged in the beam path of the overview illumination apparatus in said figure, said mask bringing about a half-sided coverage of the overview illumination pupil and being able to produce a half-pupil contrast, as is described further above.

[0111] FIG. 5b depicts the coverage in the beam path of the overview illumination apparatus and of the illumination optical unit 9, and also the resulting masking in the beam path of the detection objective 3.

[0112] Each of the exemplary embodiments described above may comprise a control unit 13 (only depicted in FIG. 5a) which may be embodied to actuate an illumination source (not depicted in any more detail), the illumination objective 2, the detection objective 3 and/or the illumination optical unit 9. Further, the control unit 13 may be embodied to evaluate the captured overview images and/or the images of the light sheet 6. The control unit 13 may be connected to a display for graphically illustrating the captured overview images and/or the images of the light sheet 6.

[0113] Different options of image acquisition are realizable, as a matter of principle, by means of one of the embodiments of the microscope 1 according to the invention. Some of the options are elucidated using the example of FIG. 6.

[0114] The simplified illustration of the light sheet 6 simultaneously specifies an object plane of the detection objective 3. A specimen scan may be carried out in the direction of the specimen plane 4 by way of a first scanning movement SB1. In so doing, the specimen volume edged by a solid line in an exemplary manner and shown as a rectangle for a simplified illustration is scanned.

[0115] In a further option, a relative motion is brought about as a second scanning movement SB2 between the specimen 5 and the light sheet 6 or the object plane in the direction of the second optical axis A2. In so doing, e.g. the specimen volume shown edged by means of an interrupted solid line is scanned.

[0116] A third option consists of producing a relative movement as third scanning movement SB3 in the direction of the third optical axis A3, within the scope of which a specimen volume shown edged by means of a dotted line is scanned.

[0117] Further, combinations of the scanning movements SB1, SB2 and/or SB3 are also possible.

[0118] The scanned specimen volumes may subsequently be transformed into Z-stacks by means of the transformation explained below.

[0119] Combinations of the exemplary embodiments within the scope of considerations by a person skilled in the art are possible.

[0120] The method according to the invention may be carried out by any one of the aforementioned embodiments of the microscope 1.

[0121] Configurations of the method are described below on the basis of the figures, in particular FIGS. 7a, 7b and 8a, 8b and 8c.

[0122] FIG. 7a shows a Z-stack in a schematic and, for reasons of presentability, slightly perspective manner, as could be realized in a coordinate system of the detection objective 3 or of a detector (not depicted here). The spacings between captured individual images EB in the direction of the Z-axis Z are denoted by Δz′ (see below).

[0123] FIG. 7b shows a Z-stack in a coordinate system of the specimen 5, in a lateral view which has been rotated in relation to FIG. 7a.

[0124] FIG. 8a depicts a virtual lattice in an exemplary and schematic manner, the X-axis X and Y-axis Y of said lattice lying parallel to the specimen plane 4 and the Z-axis Z being aligned perpendicular to the specimen plane 4. Captured individual images EB, which form a displaced Z-stack, are depicted schematically.

[0125] FIG. 8b schematically illustrates a transformation 1 described in more detail below and FIG. 8c illustrates a transformation 2.

[0126] An individual image EB (FIGS. 7a and 7b) is initially obtained in the method, said individual image having been recorded using one of the above-described embodiments of the microscope 1. The individual image EB is aligned perpendicular to the second optical axis A2 of the detection objective 3 (FIG. 7a) and at an angle of α.sub.2 in relation to the specimen plane 4 (FIG. 7b). A number of individual images EB are captured at recording positions, with the recording positions each being spaced apart from one another by a predetermined or selectable increment (denoted by Δz′). By moving (scanning) the specimen 5 in the X-direction X and recording an individual image EB at each recording position, it is possible to create a Z-stack (3D-volume stack) which, in the present case, is a displaced Z-stack since the individual images EB are aligned at an angle of α.sub.2 in relation to the specimen plane 4 (FIG. 7b).

[0127] A Z-stack is a sequence of individual images EB which lie in succession in the direction of the Z-axis Z. This may readily be carried out for specimens 5 without a preferred direction.

[0128] A specimen 5 has a preferred direction if the specimen 5 does not have any arbitrary position and/or extent in space, for example on account of external and/or actual circumstances. A cell may be mentioned in an exemplary manner, said cell lying or growing on a substrate, for example a cover slip. On account of the substrate, the shape of the cell along the contact area thereof with the substrate is predetermined and it has a substantially flat embodiment. Therefore, the cell has a preferred direction pointing away from the substrate.

[0129] Particularly in the case of specimens 5 with a preferred direction, which e.g. grow on a cover slip as a substrate, it is helpful for a user if said user is provided with a normalized, non-displaced Z-stack, as is conventional in e.g. laser scanning microscopy (LSM) or when using a rotatable pinhole aperture (spinning disk). The individual planes of a corresponding Z-stack are aligned parallel to the specimen plane 4 (XY-planes). Hence, the captured Z-stack must be converted by way of a suitable transformation (“deskew”) into a normalized, non-displaced Z-stack, the XY-planes of which are aligned parallel to the specimen plane 4. By way of example, this may be achieved by one of the two following transformations:

[0130] Transformation 1 (xyz-Interpolation)

[0131] The originally captured Z-stack is surrounded by a lattice (FIG. 8a), the X-axis X and Y-axis Y of said lattice lying parallel to the specimen plane 4 and the Z-axis Z being aligned perpendicular to the specimen plane 4. The spacings Δz′ of the individual planes may be selected in such a way that they correspond to the lateral resolution of the original Z-stack in order to obtain an isotropic voxel dimension:

Δx′=Δx

Δy′=Δy

Δz′=Δx or Δy

[0132] The calculation of the intensities at the new lattice points P.sub.x,y,z is carried out by three weighted interpolations of adjacent lattice points P.sub.x′,y′,z′ of the original lattice. The paths subjected to the interpolations are denoted by IP1, IP2 and IP3.

[0133] Transformation 2 (y-Interpolation)

[0134] The originally captured Z-stack is virtually surrounded by a lattice (FIG. 8a), the X-axis X and Y-axis Y of said lattice lying parallel to the specimen plane 4 and the Z-axis Z being aligned perpendicular to the specimen plane 4. The spacings Δz′ of the individual lattice planes may be selected in such a way that

Δx′=Δx

Δy′=Δy

Δz′=Δy*sin(α.sub.1)

applies. The calculation of the intensities at the new lattice points P.sub.x,y,z is carried out by a weighted interpolation of adjacent points P.sub.x′,y′,z′ of the original lattice (FIG. 8c).

[0135] After one of the two transformations 1 or 2 has been carried out, the transformed and, as a consequence thereof, normalized Z-stack with XY-planes lying parallel to the specimen plane 4 is available.

[0136] The recording speed achievable by means of the method may be increased further by way of one of the four following options A to D, or by way of a combination thereof.

[0137] A. A disadvantage of producing an overview image parallel to the specimen plane 4 is that it is always necessary to record a complete Z-stack. As a consequence, the production of an overview image may require a relatively long period of time. The waiting period may be reduced by virtue of increasing the increment Δz between two XY-planes and hence having to record few images.

[0138] B. Alternatively, a relatively long waiting period may also be accepted in order to record a large volume of the specimen 5 at a low resolution (e.g. including tiling). Subsequently, the entire volume is observed virtually with the aid of a 3D-viewer and a region of interest (ROI) is identified. Following this, an overview image of this region of interest may be recorded with an increased resolution, for example with smaller increments Δz between the XY-planes.

[0139] C. A further acceleration of the method may be obtained if only one XY-plane is calculated parallel to the specimen plane 4 and displayed immediately, instead of calculating the entire Z-stack at once. All interpolation may be dispensed with.

[0140] D. Alternatively, a single line may also be read directly by selecting a single-line ROI on a detector, e.g. a camera. Instead of recording many large individual images EB in the Z-direction Z, many lines are now recorded in the Z-direction Z. This may be carried out much more quickly than recording a complete individual image EB.

[0141] In methods C and D, it is possible to select which line—and hence which XY-plane parallel to the specimen plane 4—is displayed. When displaying, it must be observed that each individual XY-plane must be displaced by

[00001]$\Delta =\frac{\Delta .Math..Math.z}{\tan \left({\alpha }_1\right)}$

in relation to the previous XY-plane.

[0142] While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.

REFERENCE SIGNS

[0143] 1 Microscope

[0144] 2 Illumination objective

[0145] 20 Wide-field objective

[0146] 3 Detection objective

[0147] 4 Specimen plane

[0148] 5 Specimen

[0149] 6 Light sheet

[0150] 7 Specimen holder

[0151] 8 Liquid

[0152] 9 Illumination optical unit

[0153] B Reference axis

[0154] 10 Polarizer, Hoffmann optical unit, DIC prism

[0155] 11 Mask

[0156] 12 Diffuser

[0157] 13 Control unit

[0158] EB Individual image

[0159] NA1 Numerical aperture (of the illumination optical unit 9)

[0160] NA2 Numerical aperture (of the detection objective 3)

[0161] NA1mask Masked region (of NA1)

[0162] NA1unmask Unmasked region (of NA1)

[0163] A1 First optical axis

[0164] A2 Second optical axis

[0165] A3 Third optical axis

[0166] IP1 First interpolation

[0167] IP2 Second interpolation

[0168] IP3 Third interpolation

[0169] α1 Angle (between first optical axis A1 and third optical axis A3)

[0170] α2 Angle (between second optical axis A2 and third optical axis A3)