Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy.
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.
Nanophotonics and Nanophysics Group, H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK.
Benjamin T
BT
Miles
Nanophotonics and Nanophysics Group, H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK.
Alexander B
AB
Greenwood
School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
David
D
Benito-Alifonso
Bristol Centre for Functional Nanomaterials, H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK.|School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
Hugh
H
Tanner
School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
M Carmen
MC
Galan
Wolfson Bioimaging Facility, University of Bristol, Bristol, BS8 1TD, UK.|School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
Paul
P
Verkade
Nanophotonics and Nanophysics Group, H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK.|Bristol Centre for Functional Nanomaterials, H.H. Wills Physics Laboratory, University of Bristol, Bristol, BS8 1TL, UK.
Henkjan
H
Gersen