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.

1 . A Method for microscopic three-dimensional imaging of a sample in which image information from different depth planes of the sample is acquired and is stored in a spatially resolved manner so as to be correlated to its spatial coordinates X, Y, Z, and in which the three-dimensional image of the sample is reconstructed electronically from the stored image information, comprising the steps of:
 positioning at least one reference object is next to or in the sample;   changing the detection direction from which the image information is acquired relative to the sample and relative to the reference object;   acquiring image information from the reference object from each detection direction given by a change;   storing the acquired image information in a spatially resolved manner in spatial coordinates X, Y, Z;   acquiring image information from different depth planes of the sample from each detection direction given by a change;   storing the image information acquired from different planes in a spatially resolved manner in spatial coordinates X, Y, Z;   obtaining transformation operators for translation, rotation and deformation based on the spatial coordinates X, Y, Z of the stored image information from the reference object so that image information from the same source from the different detection directions can be overlapped; and   reconstructing a three-dimensional image of the sample from the spatial coordinates X, Y, Z of the image information obtained from the sample based on these transformation operators.   
   
   
       2 . The Method according to  claim 1 ,
 wherein the reference object is a pin, a needle, a fiber, a thread, or small particles.   
   
   
       3 . The Method according to  claim 1 , wherein the structure of the reference object is used as the reference structure, and the spatial coordinates X, Y, Z which are acquired on the basis of the reference structure and stored are used to obtain transformation operators. 
   
   
       4 . The Method according to  claim 1 ,
 wherein the transformation operators are related to their respective spatial coordinates X, Y, Z in the form of a rule for translation, rotation and deformation of the image information which is acquired from the individual detection directions and stored, and   wherein the rule is used in the reconstruction of the three-dimensional image of the sample.   
   
   
       5 . A Method for microscopic three-dimensional imaging of a sample in which image information from different depth planes of the sample is acquired and is stored in a spatially resolved manner so as to be correlated to its spatial coordinates X, Y, Z, and in which the three-dimensional image of the sample is reconstructed electronically from the stored image information, comprising the steps of: obtaining a three-dimensional image of a sample comprising a fluorescing substance or of a sample to which a fluorescing substance is added by selective plane illumination microscopy;
 illuminating the sample by an excitation beam which is shaped as a light sheet;   obtaining an image information of the sample based on the emission radiation coming from the sample,   positioning at least one reference object comprising a fluorescing substance or a reference object having a fluorescing substance next to or in the sample;   acquiring the detection direction from which the image information; acquired   changing the detection direction relative to the sample and relative to the reference object,   acquiring image information from the reference object from each detection direction given by a change;   storing the image information from the reference object from each detection direction given by a change in a spatially resolved manner in spatial coordinates X, Y, Z;   acquiring image information from different depth planes of the sample from each detection direction given by a change;   storing the image information from different depth planes in a spatially resolved manner in spatial coordinates X, Y, Z;   obtaining transformation operators for translation, rotation and deformation based on the spatial coordinates X, Y, Z of the stored image information from the reference object so that image information from the same source from the different detection directions can be overlapped;   reconstructing the three-dimensional image of the sample from the spatial coordinates X, Y, Z of the image information obtained from the sample based on these transformation operators.   
   
   
       6 . The Method according to  claim 5 , further comprising the steps of:
 applying a reference structure in the form of an irregular intensity distribution to the illumination light;   recording and evaluating images of the reference structure of the illumination light, of the reference structure of the reference object, or of a sample structure which is suitable as a reference structure prior in time to the acquisition of the image information of the reference object and of the sample from at least one of the given detection directions, with respect to the sharpness of their imaging, and   displacing the light sheet in the detection direction into the position in which the sharpest imaging of the reference structure is achieved in an observation plane, and/or the light sheet is oriented in such a way that the reference structure is sharply imaged in a homogeneous manner over the entire image field.   
   
   
       7 . The Method according to  claim 5 ,
 wherein the image information of the reference object and the image information of the sample are acquired based on the emission radiation whose wavelengths differ from one another.   
   
   
       8 . The Method according to  claim 5 ,
 wherein the transformation operators are related to their respective spatial coordinates X, Y, Z in the form of a rule for translation, rotation and deformation of the image information which is acquired from the individual detection directions and stored, and   wherein the rule is used in the reconstruction of the three-dimensional image of the sample.   
   
   
       9 . The Method according to  claim 1 ,
 wherein the image information of the sample and the image information of the at least one reference object are acquired simultaneously or successively in time.   
   
   
       10 . Method according to  claim 1 ,
 wherein the detection direction is changed by rotating the sample by 90° in each instance,   wherein the axis of rotation is oriented perpendicular to the detection directions.   
   
   
       11 . The Method for orientation of an illumination light shaped as a light sheet relative to a focus plane of a microscope objective in three-dimensional imaging of a sample using a method of selective plane illumination microscopy (SPIM), comprising the steps of:
 applying a reference structure in the form of an inhomogeneous intensity distribution to the illumination light;   positioning at least one reference object which is made of a fluorescing substance or has a fluorescing substance and which has a structure that is usable as a reference structure next to or in the sampler;   determining a characteristic structure of the sample as reference structure;   recording images of the reference structure of the illumination light, of the reference structure of the reference object or of the reference structure of the sample from at least one given detection direction;   evaluating the images of the reference structure of the illumination light, of the reference structure of the reference object or of the reference structure of the sample with respect to the sharpness of their image;   displacing the light sheet in the at least one given detection direction into a position in which the sharpest imaging of the reference structure is achieved in an observation plane; and,   orienting the light sheet in a way that the reference structure is imaged in a uniformly sharp manner over the entire image field.   
   
   
       12 . The Method according to  claim 6 , further comprising the steps of:
 moving the orientation of the light sheet in a way that that the light sheet is moved through the sample space in a z-direction;   recording an image stack during the moving step; and   evaluating the image stack with respect to the contrast of each of a plurality of individual images of the image stack,   wherein the optimal light sheet position relative to the focus plane of the microscope objective is determined based on the contrast, and the light sheet is moved into this position by means of an adjusting device.   
   
   
       13 . The Method according to  claim 5 ,
 wherein the orientation of the light sheet is carried out automatically based on the results of the evaluation of the contrast of individual images of the image stack.   
   
   
       14 . The Method according to  claim 5 ,
 wherein an object, comprised at least partially of fluorescing material, is used as reference object.   
   
   
       15 . The Method according to  claim 5 ,
 wherein a plurality of reference objects in the form of fluorescing particles, are used.   
   
   
       16 . The Method according to  claim 15 ,
 wherein at least three fluorescing particles in the form of small polystyrene beads which are marked with a dye and have a diameter in the range of 100 nm to 1 μm are used and are embedded together with the sample in a.   
   
   
       17 . The Method according to  claim 5 ,
 wherein the sample is dyed with enhanced green fluorescent protein causing the sample to fluoresce green when excited.   
   
   
       18 . The Method according to  claim 5 ,
 wherein the sample and the at least one reference object are illuminated by an excitation beam with a wavelength of 488 nm.