Selective/single plane illumination microscopy (SPIM) arrangement

Abstract

A selective/single plane illumination microscopy (SPIM) arrangement having an illumination device (1) for generating a light sheet (3) illuminating a sample (2); and a detection device (5), comprising a detector (4), for detected light proceeding from the sample (2), is configured and refined in the interest of efficient and low-impact sample investigation with physically simple means in such a way that the detection device (5) comprises a device (6) for allocating different focal planes of the light sheet (3) to different regions (7) of the detector (4).

Claims

1. A selective/single plane illumination microscopy (SPIM) arrangement, comprising: an illumination device for generating a light sheet illuminating a sample, wherein the light sheet is generated with the aid of a scanning device; and a detection device, comprising a detector for detecting light proceeding from the sample, wherein the detection device further comprises an allocation device, without a chromatic correction grating (CCG) or a prism for dispersion compensation, for allocating different focal planes of the light sheet to different regions of the detector, and wherein said allocation device is arranged in a pupil plane of a detection objective as a plug-in module in a vicinity of the detection objective, or said allocation device is attached into a lens located in a vicinity of an entrance pupil of the detection objective, and wherein a spectrum of the detected light from the sample is generated on the detector and wherein said spectrum is analyzed with predetermined software.

2. The SPIM arrangement according to claim 1, wherein the allocation device comprises a multi-focus grating (MFG).

3. The SPIM arrangement according to claim 1, wherein the detector comprises a planar detection region.

4. The SPIM arrangement according to claim 1, wherein the detector comprises a CCD, cMOS, or sCMOS.

5. The SPIM arrangement according to claim 1, wherein the detected light emerging from the allocation device is conveyed directly onto the detector via a further lens without passing through a chromatic correction grating (CCG).

6. The SPIM arrangement according to claim 1, wherein the SPIM arrangement is embodied to carry out simultaneous three-dimensional fluorescence correlation spectroscopy (3D FCS) measurements.

7. The SPIM arrangement according to claim 1, wherein the SPIM arrangement is embodied to carry out measurements based on multi-photon excitations.

8. The SPIM arrangement according to claim 1, wherein the detected light emerging from the allocation device is conveyed directly onto the detector via a further lens without passing through a prism.

9. The SPIM arrangement according to claim 1, wherein the SPIM arrangement is embodied to carry out three-dimensional fluorescence lifetime microscopy (3D FLIM) measurements.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In conjunction with the explanation of the preferred exemplifying embodiments of the invention with reference to the drawings, an explanation will also be given of generally preferred embodiments and further developments of the teaching. In the drawings:

(2) FIG. 1 is a schematic side view of an exemplifying embodiment of the SPIM arrangement according to the present invention;

(3) FIG. 2 is a schematic side view and a schematic plan view of the detection region of the exemplifying embodiment of FIG. 1, minor elements 18 having been omitted for the sake of simplicity;

(4) FIG. 3 is a schematic side view of an exemplifying embodiment of the SPIM arrangement according to the present invention, an MFG being arranged directly in the pupil plane of a detection objective;

(5) FIG. 4 is a schematic side view of a further exemplifying embodiment of the SPIM arrangement according to the present invention, a CCG and a prism additionally being omitted as compared with the exemplifying embodiment shown in FIG. 3, for which purpose the region of FIG. 4 on the right shows substantially the overall configuration of the SPIM arrangement and the region of FIG. 4 on the left shows the configuration after the detection objective as an enlarged depiction;

(6) FIG. 5 is a schematic depiction of a spectral division in the context of the exemplifying embodiment without a CCG and without a prism, in three Z planes; and

(7) FIG. 6 is a schematic side view of a conventional SPIM arrangement.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(8) FIG. 1 is a schematic side view of an exemplifying embodiment of a SPIM arrangement according to the present invention, having an illumination device 1 for generating a light sheet 3 illuminating a sample 2; and a detection device 5, comprising a detector 4, for detected light proceeding from sample 2. In the interest of efficient and low-impact sample investigation with physically simple means, detection device 5 comprises a device 6 for allocating different focal planes of light sheet 3 to different regions 7 of detector 4. Concretely, device 6 comprises an MFG 8 that is arranged in a Fourier plane.

(9) Because illumination occurs in the SPIM arrangement via an additional illumination objective 17, the pupil plane of a detection objective 19 can be used directly, as shown in FIG. 3, as a location for introducing MFG 8. This decreases the complexity of the optical configuration, and correspondingly also reduces the costs of the configuration. Greater stability can also be expected as a result of the very short beam path.

(10) MFG 8 can be arranged as a plug-in module in the immediate vicinity of detection objective 19. The entrance pupil is typically located in the vicinity of a screw-in thread of detection objective 19. It is also conceivable to incorporate MFG 8 into detection objective 19, or to attach it into a lens that is located in the vicinity of the entrance pupil.

(11) In order to compensate for a wavelength dispersion in the context of non-monochromatic detected light, device 6 comprises a suitable means 9 having a CCG 10 and a prism 11. Detected light from different focal planes passes through device 6 and strikes a planar detection region 12 of detector 4.

(12) The exemplifying embodiment of FIG. 1 comprises on the illumination side, after illumination device 1 (which is usually embodied as a laser), a beam splitter 13 that reflects illumination light onto a scanning device 14 in the form of an X-Y scanner. Illumination light then passes through a scanning lens 15 and a tube lens 16, and travels into illumination objective 17. In order to generate light sheet 3, two mirror elements 18 are arranged on detection objective 19 and generate light sheet 3 together with scanning device 14. Mirror elements 18 are arranged in such a way that an illumination beam generated by illumination device 1 can be guided via scanning device 14 and through illumination objective 17 onto mirror elements 18 in order to generate, by reflection at mirror elements 18, a substantially horizontal illumination beam (light sheet 3) for lateral illumination of sample 2.

(13) The illumination beam can be guided with the aid of scanning device 14 at the edge of the scanning field constituted by scanning device 14, the illumination beam being guided via illumination objective 17 onto mirror elements 18. Light sheet 3 is built up sequentially by moving the illumination beam along a line by means of scanning device 14. When the illumination beam is moved by means of scanning device 14 to the other side of the scanning field, the illumination beam can then strike the other mirror element 18, which then builds up a light sheet from that side. This consequently makes possible light-sheet illumination of sample 2 from several sides, as a function of the individual arrangement of mirror elements 18.

(14) Detected light proceeding from sample 2 travels through detection objective 19 to a tube lens 20 and to a further lens 21 in order to generate the Fourier plane in which MFG 8 is positioned. After passing through CCG 10 and prism 11, the detected light passes through a further lens 22 in order to focus detected light onto regions 7 on the planar detection region 12.

(15) Light sheet 3 can be generated via illumination objectives that are arranged at a 90-degree angle to an optical axis. Alternatively thereto, light sheet 3 can be generated by way of mirror elements 18 that are controlled by illumination objective 17, the illumination light beam being arranged parallel to the detection beam path. Light sheet 3 can furthermore be generated via scanning device 14 or by way of a cylindrical lens arrangement. If light sheet 3 is generated via scanning device 14, the color dispersion can advantageously be utilized. If light sheet 3 is generated by way of a cylindrical lens arrangement, the color dispersion must be compensated for by a color correction apparatus, for example CCG 10.

(16) In the context of illumination via scanning device 14, the following situation results:

(17) The illumination focus extends over a certain length and width, and forms the excitation volume. A quantity X thereof in the X direction and a quantity Y thereof in the Y direction is imaged in detection objective 19. Depending on the dimension of the illumination focus in the Z direction, the volume can encompass several imaging depths of detection objective 19. Three planes (Z1, Z0, and Z-1) will be assumed below. This situation is depicted in FIG. 2, the upper part of FIG. 2 being a side view and the lower part of FIG. 2 a plan view looking through detection objective 19 onto sample 1.

(18) When light sheet 3 is generated via scanning device 14, only a narrow region inside sample 2 is illuminated at time t0. When a camera, or detector 4, acquires an image at time t0, then only a narrow region (a few rows) on the camera chip or on detector 4 is exposed. If the different focal planes are then offset laterally on the camera chip, then depending on the number n of divisions, a corresponding number n of narrow regions is imaged simultaneously at time t0. In the exemplifying embodiment, three planes are depicted. If the camera, or detector 4, is read out synchronously with the motion of scanning device 14, for example one camera image for each illuminated region, an overall image can then be created therefrom.

(19) If the dispersion compensation resulting from CCG 10 and prism 11 is taken away (as shown in FIG. 4), a spectrum is generated on the camera chip or on detector 4 for each row. This spectrum can be analyzed using software. Changes over time in the spectrum on the scale of microseconds to milliseconds can be detected, in accordance with the image acquisition time of the camera or of detector 4 and the sweep time of scanning device 14; this can be very informative for biological processes.

(20) The configuration is appreciably simpler when MFG 8 is arranged directly in the pupil plane of detection objective 19. Omission of the unit made up of CCG 10 and prism 11 allows the dispersion of MFG 8 to be utilized, by detecting the color division as n lateral offsets on detector 4.

(21) Because a spectrum is produced on the planar detector or on detector 4 as a result of the omission of CCG 10 and prism 11, it is easily possible to analyze that spectrum in spatially resolved fashion.

(22) The mounting of MFG 8 in the pupil plane of detection objective 19 constitutes a very substantial advantage of the configuration according to the present invention, in which context additional lenses such as those necessary in the existing art can be omitted. In addition, further omission of CCG 10 and of prism 11 makes the configuration even simpler, and new capabilities are created, for example in terms of spectral detection.

(23) FIG. 5 is a schematic depiction of the spectral division of the detected light into the upper and lower Z planes in the region of detector 4, in the context of the exemplifying embodiment having no CCG 10 or prism 11. Wavelengths increase from .sub.1 to .sub.5. No such division takes place in the center plane Z.sub.0.

(24) MFG 8 can be configured in various ways. There are types based on reflection (reflection grating), transmission (transmission grating), or a prism-type embodiment (grating prisms).

(25) FIG. 6 is a schematic side view showing a conventional SPIM arrangement in which a detected light proceeding from sample 2 is guided through detection objective 19 and tube lens 20 directly onto detector 4. The conventional configuration of the SPIM arrangement according to FIG. 6 otherwise corresponds to the exemplifying embodiment according to FIG. 1, identical elements being labeled with identical reference characters.

(26) To avoid repetition, the reader is referred to the general portion of the description, and to the appended claims, with regard to further advantageous embodiments of the SPIM arrangement according to the present invention.

(27) Lastly, be it noted expressly that the exemplifying embodiment described above serves only for discussion of the teaching that is claimed, but does not limit it to the exemplifying embodiment.

PARTS LIST

(28) 1 Illumination device 2 Sample 3 Light sheet 4 Detector 5 Detection device 6 Device 7 Region 8 MFG 9 Means for compensating for a wavelength dispersion 10 CCG 11 Prism 12 Detection region 13 Beam splitter 14 Scanning device 15 Scanning lens 16 Tube lens 17 Illumination objective 18 Mirror element 19 Detection objective 20 Tube lens 21 Lens 22 Lens