title: Tracing nanoparticles and photosensitizing molecules at transmission electron microscopy by diaminobenzidine photo-oxidation.
abstract: During the last three decades, diaminobenzidine photo-oxidation has been applied in a variety of studies to correlate light and electron microscopy. Actually, when a fluorophore is excited by light, it can induce the oxidation of diaminobenzidine into an electron-dense osmiophilic product, which precipitates in close proximity to the fluorophore, thereby allowing its ultrastructural detection. This method has very recently been developed for two innovative applications: tracking the fate of fluorescently labeled nanoparticles in single cells, and detecting the subcellular location of photo-active molecules suitable for photodynamic therapy. These studies established that the cytochemical procedures exploiting diaminobenzidine photo-oxidation represent a reliable tool for detecting, inside the cells, with high sensitivity fluorescing molecules. These procedures are trustworthy even if the fluorescing molecules are present in very low amounts, either inside membrane-bounded organelles, or at the surface of the plasma membrane, or free in the cytosol. In particular, diaminobenzidine photo-oxidation allowed elucidating the mechanisms responsible for nanoparticles internalization in neuronal cells and for their escape from lysosomal degradation. As for the photo-active molecules, their subcellular distribution at the ultrastructural level provided direct evidence for the lethal multiorganelle photo-damage occurring after cell photo-sensitization. In addition, DAB photo-oxidized samples are suitable for the ultrastructural detection of organelle-specific molecules by post-embedding gold immunolabeling.
authors: Department of Neurological and Movement Sciences (Anatomy and Histology Section), University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. Electronic address: manuela.malatesta@univr.it.
authors: M
authors: M
authors: Malatesta
authors: Department of Biology and Biotechnology "Lazzaro Spallanzani" (Laboratory of Cell Biology and Neurobiology), University of Pavia, Via A. Ferrata, 9, 27100 Pavia, Italy. Electronic address: pelli@unipv.it.
authors: C
authors: C
authors: Pellicciari
authors: Department of Neurological and Movement Sciences (Anatomy and Histology Section), University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. Electronic address: bacys79@gmail.com.
authors: B
authors: B
authors: Cisterna
authors: Department of Neurological and Movement Sciences (Anatomy and Histology Section), University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. Electronic address: manuela.costanzo@univr.it.
authors: M
authors: M
authors: Costanzo
authors: Department of Biology and Biotechnology "Lazzaro Spallanzani" (Laboratory of Cell Biology and Neurobiology), University of Pavia, Via A. Ferrata, 9, 27100 Pavia, Italy. Electronic address: valentina.galimberti01@universitadipavia.it.
authors: V
authors: V
authors: Galimberti
authors: Department of Biology and Biotechnology "Lazzaro Spallanzani" (Laboratory of Cell Biology and Neurobiology), University of Pavia, Via A. Ferrata, 9, 27100 Pavia, Italy. Electronic address: marcobig@unipv.it.
authors: M
authors: M
authors: Biggiogera
authors: Department of Neurological and Movement Sciences (Anatomy and Histology Section), University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. Electronic address: carlo.zancanaro@univr.it.
authors: C
authors: C
authors: Zancanaro
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000378:metabolism*; D020811:Biomedical Technology; D045744:Cell Line, Tumor; D003600:Cytosol / Q000737:chemistry / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry*; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure; D010778:Photochemotherapy / Q000379:methods; D017319:Photosensitizing Agents / Q000737:chemistry*
chemical_list: D017319:Photosensitizing Agents; D006631:Amine Oxidase (Copper-Containing)
keywords: Diaminobenzidine photo-oxidation; Fluorogenic substrates; Nanoparticles; Photosensitizers