Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy
| Autor: | Benjamin T. Miles, Hugh Tanner, Alexander B. Greenwood, M. Carmen Galan, Paul Verkade, Henkjan Gersen, David Benito-Alifonso |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
0301 basic medicine
Correlative single particle detection Colocalisation Microscope Materials science Nanoparticle 02 engineering and technology Article law.invention 03 medical and health sciences law Correlative Light Electron Microscopy Microscopy Gold nanoparticles Multidisciplinary Cross-Polarization Imaging Scattering 021001 nanoscience & nanotechnology Fluorescence Fluorescent Labels single molecule detection 030104 developmental biology Colloidal gold Fluorescent Quenching Biophysics Electron microscope 0210 nano-technology |
| Zdroj: | Scientific Reports Miles, B T, Greenwood, A B, Benito-Alifonso, D, Tanner, H, Galan, M C, Verkade, P & Gersen, H 2017, ' Direct evidence of lack of colocalisation of fluorescently labelled gold labels used in correlative light electron microscopy ', Scientific Reports, vol. 7, 44666 . https://doi.org/10.1038/srep44666 |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/srep44666 |
| Popis: | Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field. |
| Databáze: | OpenAIRE |
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