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Cathodoluminescence (CL) and correlative light‐electron microscopy (CLEM) are two useful analytical tools in diverse research areas. Recently, fluorescent nanodiamonds (FNDs) have emerged as promising imaging agents for both CL and CLEM owing to their exceptional photophysical and chemical properties. However, to realize their practical applications in the life sciences, surface modification and functionalization of the nanomaterials with bioactive molecules are critical and essential. Here we provide a comprehensive review on the methods of synthesizing biohybrid FNDs as well as recent advances of CL and CLEM imaging of cells with these carbon nanoparticles as dual‐contrast markers. [ABSTRACT FROM AUTHOR]

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ~50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens. [ABSTRACT FROM AUTHOR]

Measurements of the photon second-order correlation function, g(2)( τ), is a common tool for the characterization of single photon emitters, like nitrogen-vacancy color centers in diamond. Such measurement requires background photoluminescence correction, which is easy when this background is homogeneous on a few wavelengths scale. However, if the sample contains emitting centers separated by a distance smaller than the optical diffraction limit, and having different and overlapping emission, these background correction techniques cannot be applied. We have recently shown that cathodoluminescence (CL) can be used to measure g(2)( τ) at the subwavelength scale. Here we propose a method, based on spatially and spectrally resolved CL, to subtract the background taking into account the nanometer spatial distribution of the emitted light. To this end, a nanometer-resolved spectrum image is acquired on the same region where the g(2)( τ) is measured. As an example, we show the use of this method to the subtraction of the H3 background signal from a g(2)( τ) measurement done on NV0 color centers. [ABSTRACT FROM AUTHOR]

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets. [ABSTRACT FROM AUTHOR]