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Contrast agent for electron correlative microscopy

Contrast agentsCorrelative microscopyelectron microscopy;

Introduction

Correlative microscopy (CLEM) is an ultrastructural analysis technique that allows obtaining orthogonal information from complementary strategies, such as fluorescence and electron microscopy. This allows gaining a simultaneous access to information regarding both structure and function of biologic specimens. The use of contrast agents based on heavy metals, however, leads to extensive fluorescence signal degradation. The contrast agent described in this invention provides high signal/noise ratios in high pressure freezing conditions, since it allows using very low contrast agent concentrations. This translates in the nearly complete elimination of fluorescence quenching. Overall, these features afford significantly improved correlative data compared with what achievable with other commonly used contrast agents employed in electron microscopy.

Technical features

Our product efficiently stains biological samples even in high pressure freezing conditions (i.e. at low concentration) providing excellent signal/noise ratio. The possibility to use these conditions, and especially the low concentrations of contrast agent needed, allows maintaining unaltered fluorescence emission of markers present in the sample. The two pictures show a fluorescent sample stained with contrasting agent described in the invention (left), highlighting that sample fluorescence is completely retained upon staining. Picture on the right shows that our product is far more efficient than uranyl acetate in contrasting a biologic sample for electron microscopy.

Possible Applications

  • Use in electron and correlative microscopy;
  • More specifically, use in those samples where it is necessary to achieve a simultaneous evaluation of function and ultrastructural localization of a given analyte.

Advantages

  • It provides excellent contrast in electron microscopy;
  • Allows using cryogenic strategies for sample preparation maintaining unaltered cell ultrastructure;
  • Allows set up of correlative electron/fluorescence microscopy protocols;
  • It is easy and cheap to acquire and dispose. It is not subject to regulatory restrictions of radioactive derivatives.