Method for examining a specimen by means of light sheet microscopy

• US 10,247,934 B2
• Filed: 11/18/2015
• Issued: 04/02/2019
• Est. Priority Date: 12/19/2014
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: Kalkbrenner, Thomas, Lippert, Helmut, Siebenmorgen, Joerg
• Primary Examiner(s): Walker, Jared
• Application Number:
  US15/537,424
• Publication Number:
  US 20180267289A1
• Time in Patent Office: 1,231 Days
• Field of Search: 348 79
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/10 affording dark-field illumination G02B21/14 and G02B21/125 take precedence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G06T 2207/10056 Microscopic image

Abstract

A method for examining a specimen via light sheet microscopy includes selecting several illumination wavelengths for the specimen. To structure the illumination light, a predefined phase distribution is impressed on the phase-selective element and a predefined aperture structure is impressed on an aperture in the aperture plane. The phase-selective element is then illuminated in an intermediate image plane in an illumination beam path with illumination light, which is structured by the phase-selective element. The structured illumination light is imaged into an aperture plane arranged downstream of the phase-selective element. The aperture structure is adapted such that the zero orders of the structured illumination light in the aperture plane are substantially removed. The specimen is illuminated with the structured light sheet in the light sheet plane. Light emitted by the specimen is detected in a detection direction which forms an angle different from zero with the light sheet plane.

Claims

1. A method for examining a specimen via light sheet microscopy, comprising:

selecting several illumination wavelengths, which comprise an illumination light for the specimen;

fixing a modulation depth of a phase-selective element at π

for a middle wavelength;

impressing a phase-selective element with a predefined phase distribution

impressing an aperture in an aperture plane with a predefined aperture structure, including;

selecting a preferred wavelength of light which comprises at least a wavelength corresponding to the longest illumination wavelength,

determining in the focal plane of an illumination objective an electric field for a light sheet of predefined shape with the preferred wavelength of light,

calculating a predefined phase distribution of the preferred wavelength of light, wherein a middle region is removed in the aperture plane, such that the aperture structure removes a zero order of the structured light of the preferred wavelength;

illuminating the phase-selective element in or close to an intermediate image plane in an illumination beam path with the illumination light;

structuring the illumination light by the phase-selective element;

imaging the structured illumination light into an aperture plane arranged downstream of the phase-selective element, in which a frequency spectrum of the illumination light is generated;

adapting the aperture structure such that the zero order of the structured illumination light in the aperture plane are substantially removed, whereby in a focal plane of the downstream illumination objective a structured, multi-coloured light sheet is formed, with a light sheet plane which is oriented perpendicular to the focal plane of the illumination objective;

illuminating the specimen with the structured light sheet in the light sheet plane; and

detecting light emitted by the specimen in a detection direction which forms an angle different from zero with the light sheet plane.

2. The method of claim 1, wherein the electric field of the light sheet is determined by coherently superimposing at least two sinc³ beams in the focal plane of the illumination objective to shape the light sheet, and the method further comprising calculating the electric field resulting in the focal plane.

3. The method of claim 1, wherein the electric field of the light sheet is determined by using Bessel beams of a predefined shape to shape the light sheet, the method further comprising determining the electric field of such a Bessel beam in the focal plane, and arithmetically determining a superimposition of the electric field of the Bessel beam and identical electric fields spaced apart from each other in each case by a predefined amount in the focal plane.

4. The method of claim 1, wherein the aperture structure removes a side lobe in the aperture plane.

5. The method of claim 1, further comprising shaping the illumination light into a beam with an intensity profile corresponding to a Gaussian distribution, which is elliptically collimated, before striking the phase-selective element.

6. The method of claim 1, wherein a diffractive optical element is used as the phase-selective element.

7. The method of claim 6, wherein a spatial light modulator is used as diffractive optical element.

8. The method of claim 1, wherein the middle wavelength is an average of a longest and a shortest of the illumination wavelengths.

9. The method of claim 1, wherein the middle wavelength is a wavelength at a highest diffraction efficiency.