Photoconversion is a correlative technique used to combine the capabilities of conventional light microscopy with the high spatial resolution and fine specific localization provided by transmission electron microscopy. In fact, photoconversion allows to translate fluorescence signals into electron-dense diaminobenzidine (DAB) deposits which are detectable in ultrastructural analysis. In this view, photoconversion may have promising application as a method for detecting fluorescently-labelled nanoparticles (NPs); in fact, the intracellular localization of NPs may elucidate the dynamic process of cell internalization, as well as their organelle targeting and final fate. Our previous study showed that photoconversion can be used to track the intracellular location of fluorescently-labeled chitosan NPs in Epon embedded samples. In this work we verify whether photoconverted samples can be suitably processed to transmission electron microscopy not only for morphological analysis but also for immunocytochemistry. Cells loaded with fluorescent NPs in vitro were fixed with aldehydes and photoconverted, and embedded in either epoxy or acrylic resins. Results demonstrate that specimens embedded in acrylic resin were of sufficiently high quality in terms of organelle preservation and definition while having much higher potential for cytochemical analyses than the samples embedded in epoxy resins for pure morphology.

Diaminobenzidine photoconversion allows detection of fluorescently-labelled nanoparticles at transmission electron microscopy after embedding in epoxy and acrylic resins

Cisterna, Barbara;COSTANZO, Manuela;MALATESTA, Manuela;ZANCANARO, Carlo
2012

Abstract

Photoconversion is a correlative technique used to combine the capabilities of conventional light microscopy with the high spatial resolution and fine specific localization provided by transmission electron microscopy. In fact, photoconversion allows to translate fluorescence signals into electron-dense diaminobenzidine (DAB) deposits which are detectable in ultrastructural analysis. In this view, photoconversion may have promising application as a method for detecting fluorescently-labelled nanoparticles (NPs); in fact, the intracellular localization of NPs may elucidate the dynamic process of cell internalization, as well as their organelle targeting and final fate. Our previous study showed that photoconversion can be used to track the intracellular location of fluorescently-labeled chitosan NPs in Epon embedded samples. In this work we verify whether photoconverted samples can be suitably processed to transmission electron microscopy not only for morphological analysis but also for immunocytochemistry. Cells loaded with fluorescent NPs in vitro were fixed with aldehydes and photoconverted, and embedded in either epoxy or acrylic resins. Results demonstrate that specimens embedded in acrylic resin were of sufficiently high quality in terms of organelle preservation and definition while having much higher potential for cytochemical analyses than the samples embedded in epoxy resins for pure morphology.
nanoparticles; photoconversion; immunohistochemistry; transmission electron microscopy
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/637553
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