Prospects and limitations of expansion microscopy in chromatin ultrastructure determination.
Chromosome Res
; 28(3-4): 355-368, 2020 12.
Article
en En
| MEDLINE
| ID: mdl-32939606
Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cromatina
/
Microscopía
Idioma:
En
Revista:
Chromosome Res
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2020
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Países Bajos