Abstract:Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and im… Show more
“…CL of FNDs for superresolution imaging has mostly been explored using single particles 5, 40 even with detecting individual defect centres within one nanodiamonds 39 . Here, we show for the first time CL of relatively small-sized FNDs within a biological context (Figs 1d,e and 3c), compared to what others have shown before 24 to achieve superresolution imaging. Bio-application of CL correlation with EM has been applied before with different nanoparticles, but size is often an issue 9, 10 .Figure 4Multimodal analysis of FND70 immunolabeling of EpCAM-GFP HT29 cells.…”
Section: Resultssupporting
confidence: 69%
“…Recently, attention has gone to relatively large FNDs 33, 34 which show high fluorescent and CL signals 24 . However, for bio-applications small FNDs, approaching the size of biomolecules, are preferred.…”
Section: Resultsmentioning
confidence: 99%
“…Fluorescence from the EM samples is however still diffraction limited, precluding precise localization when overlaid with EM data. In order to achieve high resolution localization we utilized the CL properties of the FNDs which have been shown before with single nanodiamonds and for larger FND150 in cells 5, 24, 25 . Clearly, FND40 show up in CL on a dark background (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Further interest in the FNDs stems from the fact that they can be used as local sensors of magnetic 19 or electric fields 20 , temperature 21 , or strain 22 , which could enable multi-parameter correlative microscopy. Moreover, stable CL from FNDs containing nitrogen-vacancy (NV) centers 5 , as well as silicon-vacancy centers 23 has been demonstrated, for the NV-FNDs even after cellular uptake and embedding and sectioning for scanning transmission EM 24 or in live-cell EM studies 25 . However, in these studies the FNDs are large (100–150 nm), limiting the use to cell uptake studies only.…”
Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.
“…CL of FNDs for superresolution imaging has mostly been explored using single particles 5, 40 even with detecting individual defect centres within one nanodiamonds 39 . Here, we show for the first time CL of relatively small-sized FNDs within a biological context (Figs 1d,e and 3c), compared to what others have shown before 24 to achieve superresolution imaging. Bio-application of CL correlation with EM has been applied before with different nanoparticles, but size is often an issue 9, 10 .Figure 4Multimodal analysis of FND70 immunolabeling of EpCAM-GFP HT29 cells.…”
Section: Resultssupporting
confidence: 69%
“…Recently, attention has gone to relatively large FNDs 33, 34 which show high fluorescent and CL signals 24 . However, for bio-applications small FNDs, approaching the size of biomolecules, are preferred.…”
Section: Resultsmentioning
confidence: 99%
“…Fluorescence from the EM samples is however still diffraction limited, precluding precise localization when overlaid with EM data. In order to achieve high resolution localization we utilized the CL properties of the FNDs which have been shown before with single nanodiamonds and for larger FND150 in cells 5, 24, 25 . Clearly, FND40 show up in CL on a dark background (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Further interest in the FNDs stems from the fact that they can be used as local sensors of magnetic 19 or electric fields 20 , temperature 21 , or strain 22 , which could enable multi-parameter correlative microscopy. Moreover, stable CL from FNDs containing nitrogen-vacancy (NV) centers 5 , as well as silicon-vacancy centers 23 has been demonstrated, for the NV-FNDs even after cellular uptake and embedding and sectioning for scanning transmission EM 24 or in live-cell EM studies 25 . However, in these studies the FNDs are large (100–150 nm), limiting the use to cell uptake studies only.…”
Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.
“…Existing studies of NDs using TEM have been restricted to untargeted or surface modified NDs that remain restricted to endosomes. Previous work predominantly provides isolated snapshots of NDs in cells without quantification [19], with the exception of a recent quantitative study of 150 nm NDs in endosomes [20]. To our knowledge, our TEM analysis is the first quantification of NDs with average diameter under 100 nm selectively targeted to a specific macromolecular structure and outside of endosomes.…”
Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.
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