Method for the microscopic three-dimensional reproduction of a sample

Abstract

A method for the three-dimensional imaging of a sample in which image information from different depth planes of the sample is stored in a spatially resolved manner, and the three-dimensional image of the sample is subsequently reconstructed from this stored image information is provided. A reference structure is applied to the illumination light, at least one fluorescing reference object is positioned next to or in the sample, images of the reference structure of the illumination light, of the reference object are recorded from at least one detection direction and evaluated. The light sheet is brought into an optimal position based on the results and image information of the reference object and of the sample from a plurality of detection directions is stored. Transformation operators are obtained on the basis of the stored image information and the reconstruction of the three-dimensional image of the is based on these transformation operators.

Description

[0001]The present application claims priority from PCT Patent Application No. PCT / EP2008 / 007686 filed on Sep. 16, 2008, which claims priority from German Patent Application No. 10 2007 045 897.7 filed on Sep. 26, 2007, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention is directed to a method for the three-dimensional imaging of a sample in which image information from different depth planes of the sample is acquired from at least one detection direction and is stored in a spatially resolved manner, i.e., correlated to its spatial coordinates X, Y, Z, and in which the three-dimensional image of the sample is subsequently reconstructed electronically from this image information.[0004]2. Description of Related Art[0005]It is known to obtain microscopic three-dimensional images, for example, using the principle of laser scanning microscopy (LSM). For this purpose, a pinhole diaphragm ...

AI Technical Summary

Benefits of technology

[0016]Therefore, it is the object of the invention to provide method steps which ensure an enhanced imaging quality in the three-dimensional imaging of a sample.

[0050]The advantage of using a reference structure to assist in adjusting the light sheet and in acquiring the image information consists in that the reference structure is not dependent on the sample structure and is therefore less subject, or not at all subject, to the artifacts predominantly caused by the sample itself.

[0055]In both variants of the invention, the detection direction is preferably changed by rotating the sample relative to the detection direction defined by the detection objective. The purpose of this procedure is to avoid shadow effects or to achieve a more homogeneous resolution.

[0061]The amount of image information of the reference structure is less by orders of magnitude than the amount of image information of the sample. This substantially reduces the resources required for processing.

[0074]The essential advantages of using reference structures which originate from reference objects and which can also be impressed upon the illumination light consist in that, first, a simple in-situ adjustment of the light sheet is possible when applying selective plane illumination microscopy (SPIM) and, second, the computing effort in the reconstruction of the three-dimensional image of the sample is substantially reduced compared to the prior art when reference objects are used.

Problems solved by technology

One problem with this is that measurement is not carried out directly at the sample; another is that the adjustment is carried out at a laser wavelength which does not correspond to the emission wavelength coming from the sample, which is actually the wavelength of interest, so that any chromatic longitudinal errors, e.g., of the detection objective, are not taken into account.

The adjustment of the light sheet based directly on the sample is desirable, but a suitable method for this is not known from the prior art.

Another problem with the SPIM method becomes apparent when reconstructing three-dimensional images of a sample from image information acquired from different detection directions.

The registration, i.e., the acquisition of image information and the necessary operation rules, is then very costly and time-consuming, and commercially available computers are usually not suitable for handling the required computing capacity.

Images