Method for embedding a biological sample in a transparent matrix for analysis using single plane illumination microscopy

Abstract

The invention is directed to method for positioning and aligning a preferably biological sample in the detection area of the objective of a microscope arrangement. According to the invention, the method mentioned above has the following method steps: a sample is introduced into a transparent medium, preferably agarose gel, which is initially liquid; the medium is changed from the liquid state to the solid state, wherein the sample is fixated within the medium, but the transparency of the medium is retained; the solidified medium is positioned in the microscope arrangement in such a way that the sample enclosed therein is situated in the detection area of the objective. Further, a device is proposed for positioning and aligning a preferably biological sample in the detection area of the objective of a microscope arrangement.

1 . A method for positioning a sample in the detection area of an objective in a microscope arrangement having the following method steps:
 introducing a sample into an initially liquid, transparent medium;   changing the medium from the liquid state to the solid state so that the sample is fixated inside the medium, but the medium remains substantially transparent; and   positioning the solidified medium in the microscope arrangement in such a way that the sample enclosed therein is situated in the detection area of the objective.   
     
     
         2 . The method according to  claim 1 , further comprising:
 introducing one or more samples into the transparent medium which is initially liquid;   storing the medium with the samples in a sample reservoir;   removing a partial amount of the medium with a sample contained therein from the sample reservoir;   after the removing step, changing the partial amount of medium from the liquid state to the solid state, wherein the sample is fixated within the partial amount of medium; and   positioning the cured partial amount of medium in the microscope arrangement in such a way that the sample contained therein is situated in the detection area of the objective.   
     
     
         3 . The method according to  claim 1 ;
 wherein the partial amount of medium which is still in the liquid state with the sample contained therein is sucked into the hollow space of a capillary, cannula or disposable syringe;   wherein the medium inside the hollow space is then changed from the liquid state to the solid state; and   wherein the solid medium with the enclosed sample is ejected from the hollow space.   
     
     
         4 . The method according to  claim 1 , further comprising:
 storing the transparent medium which is initially liquid on a medium reservoir;   removing a partial amount of the medium from the medium reservoir;   introducing a sample into the partial amount of medium which is still liquid;   after the removing step, changing the partial amount of medium from the liquid state to the solid state, wherein the sample ( 6 ) is fixated within the partial amount of medium; and   positioning the cured partial amount of medium in the microscope arrangement in such a way that the sample contained therein is situated in the detection area of the objective.   
     
     
         5 . The method according to  claim 4 ;
 wherein the partial amount of medium which is still liquid is introduced into the hollow space of a capillary, a cannula or a disposable syringe;   wherein the partial amount of medium which is still liquid is held inside the hollow space; and   wherein a sample is introduced into the partial amount located in the hollow space.   
     
     
         6 . The method according to  claim 5 ;
 wherein one of the two end openings of the hollow space containing the medium which is still in the liquid state is hermetically closed; and wherein the method further comprises:   introducing the sample into the medium which is still in the liquid state inside the hollow space through the opposite second end opening;   next, changing the medium inside the hollow space from the liquid state to the solid state; and   next, ejecting the solid medium with the enclosed sample ( 6 ) is ejected from the hollow space.   
     
     
         7 . The method according to  claim 1 ;
 wherein the change of the partial amount from the liquid state to the solid state is carried out under external influences on the medium.   
     
     
         8 . The method according to  claim 1 ;
 wherein a curable gel is used as medium.   
     
     
         9 . The method according to  claim 1 ;
 wherein an automation of the method steps in their entirety or an automation of some of the individual method steps is provided.   
     
     
         10 . The method according to  claim 9 ;
 wherein a first sample is introduced into the gel in the hollow space at predetermined time intervals;   wherein the gel is pushed forward;   wherein a second sample is introduced into the agarose gel;   wherein the gel is pushed forward again; and   wherein a third sample is introduced into the gel, and so on, until a given quantity n of samples has been introduced into the gel, wherein distances a are adjusted between the samples depending on the timing and forward feed speed.   
     
     
         11 . A device for introducing a sample into the detection area of an objective of a microscope arrangement, comprising:
 a reservoir for a transparent medium which is initially still liquid;   means designed for:
 removing a partial amount of the liquid medium and for introducing a sample into this partial amount; or 
 introducing samples into the liquid medium inside the reservoir and removing a partial amount of the medium with a sample contained therein; 
   means for changing the removed partial amount of medium from the liquid state to the solid state, wherein at least one sample is fixated within the partial amount of the medium; and   a device for positioning and aligning the solidified partial amount of medium in the microscope arrangement in such a way that the sample contained therein is situated in the detection area of the objective.   
     
     
         12 . The device according to  claim 11 , further comprising:
 a manipulating unit which has a hollow needle in which a suction and delivery piston is movably guided for sucking a partial amount of the medium into the hollow needle or ejecting it from the hollow needle.   
     
     
         13 . The device according to  claim 12 ;
 wherein the manipulating unit is connected to the microscope arrangement by a holder, and the holder is constructed with means for changing the position and alignment of a capillary or cannula relative to the microscope arrangement, wherein there is a change in position in coordinates X, Y, Z and a rotational movement around the longitudinal direction of the capillary or cannula by an angle φ.   
     
     
         14 . The device according to  claim 11 ;
 wherein a capillary, including the suction and delivery piston, is fastened to the manipulating unit by means of connection elements which can be disconnected manually.   
     
     
         15 . A device for embedding a plurality of samples in a curable transparent medium, comprising.
 an access for the medium, an access for the samples;   forward feed devices for the medium and the samples;   a device for introducing the samples individually into the medium successively in a timed manner until a given quantity n of samples has been inserted into the medium; and   wherein the medium is advanced in a timed manner, and distances a are adjusted between the samples within the medium depending on the timing and forward feed speed.   
     
     
         16 . The device according to  claim 12 ;
 wherein the hollow needle of the manipulating unit is in the form of a capillary or a cannula.