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Contrast solution for the characterisation of biological samples by electron or correlative microscopy

A solution and general formula technology, applied in the preparation of test samples, sampling, microlithography exposure equipment, etc.

Pending Publication Date: 2021-12-14
意大利科技研究基金会 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] The applicant has in fact discovered that it is possible to obtain contrast efficiencies not only comparable to but even better than those of standard uranyl acetate by combining lanthanide salts with heteropolyacids, which are commonly used in optical Used in microscopy, but also considered poor in electron microscopy

Method used

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  • Contrast solution for the characterisation of biological samples by electron or correlative microscopy
  • Contrast solution for the characterisation of biological samples by electron or correlative microscopy
  • Contrast solution for the characterisation of biological samples by electron or correlative microscopy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] PTA-YbCl 3 Preparation of solutions (stock solutions)

[0063] A solution of phosphotungstic acid (concentration 3.2 mM) in 10 ml of water / ethanol containing 20% ​​(v / v) ethanol was prepared. The pH of the solution was brought to about 5 with sodium hydroxide 1M. To this solution was added an equal volume of a solution of ytterbium chloride hexahydrate 48 mM (final concentration) in ethanol / water containing 20% ​​(v / v) ethanol. The pH was adjusted again to 5 with sodium hydroxide 1M. The mixture was kept under stirring at room temperature overnight. The solution thus obtained was called "X solution 1.5" and showed a precipitate. The precipitate was removed by filtration, and the pH was adjusted again to about 5 by adding a 20 mM solution of MES. The solution was then again kept under stirring overnight and filtered again, this time through a membrane with a pore size of 200 nm, in order to obtain a solution free of precipitates and referred to as "stock solution"...

Embodiment 2

[0065] Use of the solution according to the invention as a contrast medium for correlative microscopy using the HPF / FS protocol (CLEM) Characterization of Biological Samples

[0066] The stock solution (0.2 ml) obtained according to Example 1 was lyophilized and dissolved in 2 ml ("X Sol 1 / 10"), 3.2 ml ("X Sol 1 / 16") or 6 ml ("X Sol 1 / 30") ) in a water / acetone mixture containing 95% (v / v) acetone in order to obtain the solutions according to the invention in three different concentrations respectively.

[0067] Each solution was centrifuged, and the supernatant was used on a cell line stably transfected with a fluorescent protein conjugated to the endoplasmic reticulum in the standard HPF / FS protocol ( figure 1 medium in the diagram). These were used as standard samples for correlation protocols. Cells embedded in this protocol are processed by high pressure freezing / water displacement (HPF / FS) with organic solvents at cryogenic temperatures. Sample sections (cut by us...

Embodiment 3

[0071] Use of the solution according to the invention as a contrast agent for use in HPF / FS protocols by correlative microscopy (CLEM) Characterization of biological samples (Drosophila oocytes)

[0072] Experiments similar to those described in Example 2 were performed using Drosophila oocytes as biological samples and ethanol as the organic solvent instead of acetone. The images obtained are in Figure 5 shown in .

[0073] The images thus obtained show that the solutions of the invention are able to obtain comparable results in terms of fluorescence emission, while at the same time ensuring a better contrast than when using a solution comprising uranyl acetate, thus demonstrating that after correlation microscopy ( CLEM) characterization is more effective.

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Abstract

The invention relates to a solution comprising: a heteropolyacid and a lanthanide salt in a solvent consisting of a water / organic solvent mixture or an organic solvent. Optionally, the solution can also comprise an organic or inorganic buffer with a pH comprised in the interval of 4-6 or a strong base or acid. The object of the present invention also includes a use of said solution and any products of the reaction between the components thereof as a contrast medium for biological samples and a method for preparing biological samples that uses said solution in at least one step, for analysis by either correlative microscopy (CLEM) and electron microscopy (EM).

Description

technical field [0001] The present invention relates to the field of substances used for the preparation of biological samples which have to be characterized by means of electron microscopy or correlated microscopy. More precisely, the present invention relates to a contrast solution as an alternative to uranyl acetate for the characterization of biological samples by correlation microscopy (CLEM) or electron microscopy (EM). current technology [0002] In recent years, correlative microscopic characterization methods have attracted great interest in the field of diagnostics and research, above all due to the possibility of integrating information from different microscopic methods. One of the most interesting techniques is the so-called correlated light-electron microscopy (CLEM), a combination of fluorescence microscopy (FM) or light microscopy (LM) and electron microscopy. Microscopy (EM) techniques. This technique enables the extrapolation of data from a single sample ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G03F7/20
CPCG01N1/30G01N2001/302
Inventor 瓦伦蒂娜·凯佩罗乔瓦尼·西尼奥雷塞巴斯蒂亚诺·迪皮耶特罗梅丽莎·桑蒂奥尔多·莫斯卡帝尼毛罗·盖米
Owner 意大利科技研究基金会
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