Homogeneous multifocal excitation for high-throughput super-resolution imaging.
Nat Methods
; 17(7): 726-733, 2020 07.
Article
em En
| MEDLINE
| ID: mdl-32572233
ABSTRACT
Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Microscopia de Fluorescência
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article