Multimodal Imaging and Soft X‐Ray Tomography of Fluorescent Nanodiamonds in Cancer Cells

Abstract

Multimodal imaging promises to revolutionize the understanding of biological processes across scales in space and time by combining the strengths of multiple imaging techniques. Fluorescent nanodiamonds (FNDs) are biocompatible, chemically inert, provide high contrast in light‐ and electron‐based microscopy, and are versatile optical quantum sensors. Here it is demonstrated that FNDs also provide high absorption contrast in nanoscale 3D soft X‐ray tomograms with a resolution of 28 nm in all dimensions. Confocal fluorescence, atomic force, and scanning electron microscopy images of FNDs inside and on the surface of PC3 cancer cells with sub‐micrometer precision are correlated. FNDs are found inside ≈1 µm sized vesicles present in the cytoplasm, providing direct evidence of the active uptake of bare FNDs by cancer cells. Imaging artefacts are quantified and separated from changes in cell morphology caused by sample preparation. These results demonstrate the utility of FNDs in multimodal imaging, contribute to the understanding of the fate of FNDs in cells, and open up new possibilities for biological imaging and sensing across the nano‐ and microscale.

Imaging of Virus-Infected Cells with Soft X-ray Tomography

“…Cryo-SXT can provide understanding of the spatial organization of the different cellular organelles during the infection, but to achieve a better comprehension of the complexity of virus-host interactions, it is recommended to combine cryo-SXT data with other imaging techniques that can provide the precise localization or the higher resolution structure of the particular viral or host factors being studied. Correlative workflows combining cryo-SXT with techniques such as cryo-ET, visible light fluorescence microscopy, hard X-ray fluorescence tomography or numerical simulations have already proven their potential in several multi-length scale structural studies [24,[43][44][45][46][75][76][77]. A particularly powerful approach is a correlative cryo-imaging pipeline, in which the same sample is imaged with super-resolution 3D structured illumination fluorescence microscopy (cryo-SIM) and with cryo-SXT, allowing for a comprehensive view of both cellular ultrastructure and the related molecular organization over extended cellular volumes [78,79].…”

Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein–nanomaterial hybrids

“…In figure 1D a single slice from the correlated 3D datasets is shown (see S4 for the correlation accuracy map and for a video of the whole 3D stack). No laser damage was noticed at the achievable resolution of cryo-SXT (30 nm half pitch according to the FSCe/o criterion, see S5) 19,[31][32][33] after cryo-3D-SIM. Correlating both 3D datasets, the TPR-Hsp90-AuNC signal was unambiguously located in multivesicular bodies (MVB) which were homogenous in size (0.5 -1 μm in diameter) and distributed throughout the cytoplasm (Figures 1B, 1D and Figure 2).…”

The use of soft X-ray tomography to explore mitochondrial structure and function

“…Recent studies have utilized SXT to investigate different aspects of mitochondrial structure and function, demonstrating the exciting promise of SXT as a tool for interrogating the relationship between mitochondrial structure and cellular metabolism. Mitochondria have been explored by SXT in the context of healthy single cells [ 26 ] and cancer cells [ 37 , 38 ], and after isolation from cells to explore their internal structure [ 27 , 39 ]. For the purpose of this review, we will highlight four unique applications of SXT that have been used to investigate the internal structure of mitochondria [ 27 ], evaluate mitochondria-ER contacts [ 19 ], explore the interaction between mitochondria and endosome-like vesicles [ 40 ], and visualize the mitochondrial network structure during multiple phases of insulin secretion [ 16 ].…”