Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells
| Autor: | Hagen, C, Werner, S, Carregal-Romero, S, N Malhas, A, G Klupp, B, Guttmann, P, Rehbein, S, Henzler, K, C Mettenleiter, T, J Vaux, D, J Parak, W, Schneider, G, Grünewald, K |
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| Rok vydání: | 2014 |
| Předmět: |
PSS
poly(styrene sulfonate) Apoptosis NEC nuclear egress complex NR nucleoplasmic reticulum PAH poly(allylamine hydrochloride) Article cryoFM fluorescence cryo-microscopy Live-cell imaging N.A. numerical aperture Animals Nanotechnology Gold nanoparticles Nuclear membrane invaginations Instrumentation Saquinavir cryoXM/T soft X-ray cryo-microscopy/tomography Cell Nucleus Herpesvirus egress Quantum dots Cryoelectron Microscopy X-ray imaging HIV Protease Inhibitors Atomic and Molecular Physics and Optics Electronic Optical and Magnetic Materials eGFP enhanced green fluorescent protein Nanoparticles Rabbits Tomography X-Ray Computed cryoEM/T electron cryo-microscopy/tomography |
| Zdroj: | Ultramicroscopy |
| ISSN: | 0304-3991 |
| DOI: | 10.1016/j.ultramic.2014.05.009 |
| Popis: | Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. Highlights • Nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated cells. • New polyelectrolyte-Qdot® 605 coated gold beads were employed as fiducials. • Saquinavir can induce a strong apoptotic phenotype in the nucleus. • CryoXT is an auspicious imaging technique in apoptosis research. |
| Databáze: | OpenAIRE |
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