Embedding medium for biological samples, method for producing embedded biological samples, and use thereof

Abstract

In a first aspect, the invention relates to the use of a UV-polymerizable composition as an embedding medium for biological samples. The UV-polymerizable composition is a composition with a refractive index ranging from n=1.45 to n=1.6 after a polymerization process. In another aspect, the invention relates to a method for producing embedded biological samples using said UV-polymerizable composition and to the embedded biological samples themselves. The biological samples can be used in a wide variety of areas, such as diagnostics among others, and are suitable in particular for RNA-based diagnostics.

Claims

1. A method for producing an embedded biological sample, comprising the steps: a) dewatering the biological sample, b) infusing a UV-polymerizable composition into the biological sample, wherein said UV-polymerizable composition has a refractive index after polymerization in the range from n=1.45 to n=1.6, c) embedding the infused biological sample by curing the UV-polymerizable composition with light having a wavelength of 470 nm, and the UV-polymerizable composition comprises a mercapto ester compound.

2. The method as claimed in claim 1, whereby polymerization does not proceed continuously at the start.

3. The method as claimed in claim 1, wherein the biological sample was fixed before the dewatering.

4. The method as claimed in claim 1, wherein at least the infusion of the UV-polymerizable composition is carried out under vacuum.

5. The method as claimed in claim 1, wherein the dewatering of the biological sample proceeds i) by means of an ascending alcohol series or ii) by means of an ascending DMSO series.

6. The method as claimed claim 1, wherein the mercapto ester compound is present in the UV-polymerizable composition at at least 40% by weight.

7. The method as claimed in claim 1, wherein the refractive index of the UV-polymerizable composition before polymerization is in a range from n=1.4 to 1.6.

8. The method as claimed in claim 1, wherein the UV-polymerizable composition additionally comprises at least one of a polymerizable acrylate compound and a methacrylate compound.

9. The method as claimed in claim 1, wherein the biological sample is a three-dimensional biological sample and wherein said three-dimensional sample has a smallest edge length of at least 50 m.

10. The method according to claim 1 wherein said UV-polymerizable composition has a refractive index after polymerization in the range from n=1.550 to n=1.565.

11. The method according to claim 2 further comprising cooling the biological sample between incidences of light having a wavelength of 470 nm.

12. The method as claimed in claim 3 wherein the biological sample was fixed with aldehyde mixtures or alcohols.

13. The method as claimed in claim 5, wherein the dewatering of the biological sample proceeds by means of an ascending ethanol series.

14. The method according to claim 5 further comprising subsequent dewatering in xylene.

15. The method as claimed in claim 7 wherein the refractive index of the UV-polymerizable composition before polymerization is in the range from n=1.50 to 1.55.

16. The method as claimed in claim 9 wherein the biological sample is a three-dimensional biological sample and wherein said three-dimensional sample has a smallest edge length of at least 100 m.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic drawing of processing steps according to the invention;

(2) FIG. 2A is an image of a sample studied using SLOT;

(3) FIG. 2B is an image of a sample stained with Hemalaun;

(4) FIG. 2C is an RNA expression profile;

(5) FIG. 3 is a schematic drawing of processing steps for examining biological samples.

DETAILED DESCRIPTION

(6) In this case, the processing steps are shown schematically in FIG. 1:

(7) In a first step, the sample is taken and optionally fixed. The sample is then dewatered. Subsequently thereto, the embedding medium according to the invention is infused with the composition according to the invention after said composition has been appropriately produced. Subsequently, there proceeds the polymerization by means of UV irradiation to obtain a biological sample embedded according to the invention.

(8) In FIG. 3, the processing steps of the method according to the invention for examining biological samples are shown schematically. In this case, the processing steps shown in FIG. 1 are carried out. This is followed by carrying out the imaging method, for example using tomographic methods. In a further step, the production of optionally consecutive histological sections using suitable devices follows thereon.

(9) Subsequently thereto, sections obtained in such a manner are subjected to a histological examination. This histological examination can include a molecular-biological examination.

(10) In an embodiment, correlation of the data of the three-dimensional imaging, that is to say the three-dimensional model, with the histological and optionally molecular-biological data then takes place.

EXAMPLE 1

(11) The sample is taken and washed with known agents, e.g. sodium chloride solution, and then fixed. The fixing proceeds using known media, e.g. aldehyde mixtures, such as 4% of p-formaldehyde or 0.1% glutaraldehyde.

(12) The samples thus fixed are then dewatered in an ascending alcohol series and then transferred to xylene. The steps in this were as follows: 30% ethanol in distilled water, 2-4 hours, 50% ethanol in distilled water, 2-4 hours, 70% ethanol in distilled water, 2-4 hours, 90% ethanol in distilled water, overnight, 99.8% ethanol, 4 hours, 100% ethanol, 4 hours, 50% ethanol/50% xylene, overnight, 100% xylene, 4 hours, 100% xylene, 4 hours.

(13) Subsequently, xylene was mixed with the UV-polymerizable agent in a proportion of 1:1 and the sample was incubated overnight in a vacuum cabinet at <100 mbar with said 1:1 mixture. Then, the biological sample was treated with 100% UV-polymerizable composition for 6 hours in the vacuum cabinet at <100 mbar.

(14) Production of the UV-Polymerizable Sample:

(15) The UV-polymerizable sample was produced from a mixture of NOA (Norland Optical Adhesive 68, Norland Products Inc., Cranbury, USA and Norland Optical Adhesive 71, Norland Products Inc., Cranbury, USA). The mixing ratio of these two components was set in this case in such a manner that the refractive index of said UV-polymerizable composition had a value of approximately 1.523 before polymerization.

(16) A preferred mixing ratio in this case is one of 1:7 of NOA 68:NOA 71.

(17) According to the invention, a 24-hour polymerization takes place as curing of said plastic, which led to a refractive index of approximately 1.556. To measure the refractive index in the polymer, plastic blocks are polymerized parallel to the embedded samples, which blocks are of identical size to the embedded samples themselves. These plastic blocks are then, in preparation for the refractive index measurement on the refractometer (Mller Abbe Refractometer AR-4), are ground flat and finally brought onto the measuring prism with a contact medium of a higher refractive index.

(18) The polymerization can proceed continuously or batchwise.

(19) Batchwise Polymerization:

(20) The sample, for curing, was charged in syringes (fill level 3 to 5 ml, depending on sample size) together with the UV-polymerizable composition and held with an additional sample holder device. For this purpose, polymerization was started in the sample by alternating phases each of 1 minute of UV light (Leica EM AFS2) and 1 minute with cooling at 4 C. After 30 minutes, the syringe was inverted and the additional sample holder device mounted in advance was removed, and so the sample was present positioned in the center of the syringe. Subsequently, in alternating steps of UV and cooling, the polymerization was continued and finally ended by long-term irradiation with UV at room temperature.

(21) FIG. 2A-C show the detection of RNA from a human lung sample embedded according to the invention. After the embedding of the sample according to the invention, said sample was studied microscopically by means of SLOT (FIG. 2A) and then 4 m-thick sections were prepared and stained with Hemalaun (FIG. 2B). Areas of interest were cut out (excerpt B) using a laser microdissection system and incubated overnight in proteinase K buffer. Subsequently, the RNA suspended in the supernatant was purified by phenol-chloroform precipitation, and the synthesis of cDNA and amplification with suitable primers proceeded according to known methods. FIG. 2C shows an RNA expression profile having the following markers: Polyr2a (polymerase 2alpha), BMP4 (bone morphogenic protein 4), CD34, ACTA2 (alpha-2 smooth muscle-actin), COL3A1 (collagen 3A1), TIMP1 (metallopeptidase inhibitor 1), MMP2 (matix metalloprotease 2), CD14.

(22) RNA extraction, transcription and amplification of the cDNA proceeded using known methods.