Multicolour electron microscopy as never before


Rebecca Pool

Wednesday, March 27, 2019 - 13:00
Image: Professor Steven Chu and colleagues have synthesised nine nanoparticles doped with lanthanide ions for multi-colour electron microscopy [Stanford Libraries]
A team of US-based researchers led by Nobel Laureate Professor Steven Chu from Stanford has developed bright sub-20 nm coloured labels for electron microscopy.
Designed for labelling biomolecules, the luminescent nanoprobes are based on NaGdF4 or NaYF4 nanoparticles doped with one of nine lanthanide ions.
Depending on which rare earth dopant has been used, the nanoprobes emit different coloured light under electron excitation from the microscope's electron beam, via cathodoluminescence.
The method is set to provide new insights into the molecular biology of a cell. 
As Chu and colleagues write in Nature Nanotechnology, nanoscale imaging of biomolecules in the context of cellular structures is essential to understand how cells function.
But while conventional EM is used to analyse heavy-metal-stained cellular ultrastructure, such as  lipid membranes and chromatin, this method cannot implicitly provide information on biomolecule location.
And although researchers have tagged proteins with organic dyes, fluorescent proteins and quantum dots, these particles rapidly deteriorate when exposed to the electron beam.
With this in mind, the researchers set out to develop a reliable bioimaging method to localise individual proteins as well as resolve protein-protein interactions with respect to cellular ultrastructure.
As Chu and colleagues point out, stable, luminescent nanodiamonds and lanthanide-doped nanoparticles have already been used but at some 40nm in size, are too large for efficient protein labelling in biological experiments.
Given this, the researchers synthesised cathodoluminescent lanthanide-doped NaGdF4 and NaYF4 nanocrystals with diameters less than 20 nm, comparable to the quantum dots, gold nanoparticles, and immunoglobulin antibodies used to label proteins, one type at a time, in electron microscopy. 
As part of their imaging system, a SEM with a parabolic reflector was used to excite the cathodoluminescence of these lanthanide-doped nanoparticles and image the signal onto a photo-multiplying detector.
At the same time, the microscope also acquired the secondary electron signal from the same pixels registered in the cathodoluminescent (CL) channel.
To prove the nanoscale resolution capability of their method for biological applications, the researchers went on to acquire CL-SEM images of the lanthanide-doped nanoparticles.
Results indicated that by optimising nanoparticle composition, synthesis protocols and electron imaging conditions, the researchers could achieve high signal-to-noise localisation of biomolecules with a sub-20-nm resolution, limited only by the nanoparticle size.
Importantly, while imaging many nanoparticles, the luminescent labels exhibited narrow spectra of nine distinct colours, according to the dopant.
Future directions
The researchers now intend to optimise the synthesis of nanoprobes using other lanthanide ions, which they hope will pave the way to true multicolour imaging at the single-nanoparticle level.
They reckon up to nine different colours with 10-20 nm spatial resolution could potentially be achieved.
However, they also believe they can increase the number of colours further by co-doping nanoparticles with many lanthanide ions.
“Although reliable multicolour cathodoluminescence imaging at the single-nanoparticle level and in biological tissue remains to be demonstrated, our findings motivate future work in this direction,” says Chu. “Optimal multicolour imaging combined with advances in particle functionalisation and labelling could allow us to visualise the locations of different proteins with respect to the cellular ultrastructure.”
Chu and colleagues also highlight how combining multicolour cathodoluminescence imaging with recent advances in situ serial-block-face SEM or focused ion beam SEM would permit 3D reconstruction of the entire tissue sections, while providing simultaneous nanoscale protein localization.
“Such bio-specific volumetric electron imaging would enable the visualization of different cell types within heterogeneous tissue sections and shed light on the organisation of complex systems such as the heart, the brain, or cancerous tissue,” says Chu.
Research is published in Nature Nanotechnology.
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