ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life

Abstract

Nanometer-scale identification of multiple targets is crucial to understand how biomolecules regulate life. Markers, or probes, of specific biomolecules help to visualize and to identify. Electron microscopy (EM), the highest resolution imaging modality, provides ultrastructural information where several subcellular structures can be readily identified. For precise tagging of (macro)molecules, electron-dense probes, distinguishable in gray-scale EM, are being used. However, practically these genetically-encoded or immune-targeted probes are limited to three targets. In correlated microscopy, fluorescent signals are overlaid on the EM image, but typically without the nanometer-scale resolution and limited to visualization of few targets. Recently, analytical methods have become more sensitive, which has led to a renewed interest to explore these for imaging of elements and molecules in cells and tissues in EM. Here, we present the current state of nanoscale imaging of cells and tissues using energy dispersive X-ray analysis (EDX), electron energy loss spectroscopy (EELS), cathodoluminescence (CL), and touch upon secondary ion mass spectroscopy at the nanoscale (NanoSIMS). ColorEM is the term encompassing these analytical techniques the results of which are then displayed as false-color at the EM scale. We highlight how ColorEM will become a strong analytical nano-imaging tool in life science microscopy.

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

“…[115][116][117][118] "ColorEM" or analytical electron microscopy converts greyscale analytical information into false color-coded images, and can be used to identify biological features based on elemental composition. 119,120 ColorEM techniques include energy dispersive X-ray analysis, electron energy loss spectroscopy, and correlative cathodoluminescence electron microscopy, which are reviewed in great detail by Pirozzi et al 120 The interaction between an incident electron beam and the sample generates additional signals, as described previously. Elemental analysis in electron microscope can be achieved by using EDX or EELS.…”

“…All the above described methods are based on primary electron beam interaction with the sample and have a potential for label-free detection. 118,120,122 In combination with STEM, all the methods have nanometric spatial resolution and complement each other, as the resolution is mostly determined by the sample thickness and electron beam spot size. 120,130 Highenergy STEM-EELS imaging can induce significant beam damage, 132 while EDX and CL are less invasive due to the lower beam energies.…”

High-resolution synchrotron imaging studies of intact fresh roots reveal soil bacteria promoted bioremediation and bio-fortification

“…While direct chemical element localization has been done from fresh plant roots (Wang et al , 2013, Hu et al , 2014, Zhao et al , 2014, Krajcarová et al , 2017) and seeds (Rodrigues et al , 2018) using synchrotron radiation x-ray fluorescence microscopy, the resolution has not been sufficient to determine subcellular element concentrations. Using sectioning techniques and 2D imaging methods, such as nanosecondary ion mass spectrometry (nano SIMS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), or ColorEM (electron microscope) one could potentially reach desired resolution and sensitivity (Ondrasek et al , 2019, Pirozzi et al , 2018). However, these techniques can only show small fraction of the total volume and are subject to artefacts from the sectioning process.…”

Correlative Cathodoluminescence Electron Microscopy: Immunolabeling Using Rare‐Earth Element Doped Nanoparticles

“…The limited availability of protein labels suitable for CCLEM constitutes a major roadblock in the adoption of this highly promising imaging technique. [ 19 ] Most of the established fluorescence microscopy labels are unsuitable for CCLEM due to their limited electron beam stability and emission brightness. [ 18,20 ] CCLEM labels should preferably be in the size of a typical protein [ 21 ] and exhibit ultra‐bright and stable emission properties.…”