Abstract
Correlative Imaging (CI) visualizes a single sample/region of interest with two or more imaging modalities. The technique seeks to elucidate information that may not be discernible by using either of the constituent techniques in isolation. Correlative Light Electron Microscopy (CLEM) can be employed to streamline workflows, i.e., using fluorescent signals in the light microscope (LM) to inform the user of regions which should be imaged with electron microscopy (EM). The efficacy of correlative techniques requires high spatial resolution of signals from both imaging modalities. Ideally, a single point should originate from both the fluorescence and electron density. However, many of the ubiquitously used probes have a significant distance between their fluorescence and electron dense portions. Furthermore, electron dense metal nanoparticles used for EM visualization readily quench any proximal adjacent fluorophores. Therefore, accurate registration of both signals becomes difficult. Here we describe fluorescent nanoclusters in the context of a CLEM probe as they are composed of several atoms of a noble metal, in this case platinum, providing electron density. In addition, their structure confers them with fluorescence via a mechanism analogous to quantum dots. The electron dense core gives rise to the fluorescence which enables highly accurate signal registration between epifluorescence and electron imaging modalities. We provide a protocol for the synthesis of the nanoclusters with some additional techniques for their characterization and finally show how they can be used in a CLEM set up.
Original language | English |
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Pages (from-to) | 39-68 |
Number of pages | 30 |
Journal | Methods in Cell Biology |
Volume | 162 |
DOIs | |
Publication status | Published - 26 Feb 2021 |
Bibliographical note
Funding Information:Hugh Tanner was supported by the “Bristol Centre for Functional Nanomaterials,” an EPSRC-funded Doctoral Training Programme. Lorna Hodgson and Judith Mantell were funded on a BBSRC sLoLa grant (BB/M002969/1). We would like to thank the Wolfson Bioimaging Facility staff for their expert help in these studies with equipment used funded through BBSRC ALERT13 funding (BB/L014181/1).
Funding Information:
Hugh Tanner was supported by the ?Bristol Centre for Functional Nanomaterials,? an EPSRC-funded Doctoral Training Programme. Lorna Hodgson and Judith Mantell were funded on a BBSRC sLoLa grant (BB/M002969/1). We would like to thank the Wolfson Bioimaging Facility staff for their expert help in these studies with equipment used funded through BBSRC ALERT13 funding (BB/L014181/1).
Publisher Copyright:
© 2021 Elsevier Inc.
Keywords
- CLEM
- markers
- probes
- correlative microscopy
- nanoclusters