Abstract
Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. For Transmission Electron Microscopy analysis, biological samples are generally embedded in resins, which allow thin sectioning of the specimen. Embedding resins are also composed by light atoms, thus the contrast difference between the biological sample and the surrounding resin is minimal. Due to that reason in the last decades, several staining solutions and approaches, performed with heavy metal salts, have been developed with the purpose of enhancing both the intrinsic sample contrast and the differences between the sample and resin. The best staining was achieved with the uranyl acetate (UA) solution, which has been the election method for the study of morphology in biological samples. More recently several alternatives for UA have been proposed to get rid of its radiogenic issues, but to date none of these solutions has achieved efficiencies comparable to UA. In this work, we propose a different staining solution (X Solution or X SOL), characterized by lanthanide polyoxometalates (LnPOMs) as heavy atoms source, which could be used alternatively to UA in negative staining (NS), in en bloc staining, and post sectioning staining (PSS) of biological samples. Furthermore, we show an extensive chemical characterization of the LnPOM species present in the solution and the detailed work for its final formulation, which brought remarkable results, and even better performances than UA.
Highlights
Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power
We describe our results in the rational development of X Solution, an innovative staining agent based on a pH buffered Yb3+/phosphotungstic acid (PTA) mixture, and we show how, under optimized conditions, our staining agent significantly improves upon the staining efficiency of uranyl acetate (UA), in en bloc staining as well as in negative staining (NS), and post sectioning staining (PSS) procedures
We found that there is a clear influence of lanthanide salt on the signal of monobutylphosphate, which broadens upon addition of the lanthanide
Summary
Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. Biological specimens are formed by light atoms the local tiny density variations should be enhanced to give a detectable contrast in electron micrographs This is usually achieved by using heavy metals as staining a gent. This is usually achieved by using heavy metals as staining a gent1 In this field, uranyl acetate (UA) solution has been considered as the gold standard for electron microscopy (EM) characterization, due to its low cost, the very high contrast provided and its intrinsic affinity for biological s amples. The mildly radioactive nature of Uranium-derivatives raised considerable regulatory issues for their use (such as health, availability, price, storage, and disposal) These factors raised an increasing interest in developing safer, more effective alternatives to UA, and several uranium-free staining solutions have been proposed. Italiano di Tecnologia, Center for Nanotechnology Innovation @NEST, Piazza San Silvestro 12, 56127 Pisa, Italy. 6These authors contributed : Aldo Moscardini and Sebastiano Di Pietro. *email: g.signore@
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