Most cited article - PubMed ID 32939606
Prospects and limitations of expansion microscopy in chromatin ultrastructure determination
The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.
Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.
- Keywords
- Leishmania major, Trypanosoma brucei, expansion microscopy, microtubule-based cytoskeleton,
- MeSH
- Microscopy, Electron methods MeSH
- Kinetochores metabolism ultrastructure MeSH
- Kinetoplastida metabolism ultrastructure MeSH
- Leishmania major metabolism ultrastructure MeSH
- Microtubules metabolism ultrastructure MeSH
- Protozoan Proteins metabolism MeSH
- Trypanosoma brucei brucei metabolism ultrastructure MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Protozoan Proteins MeSH
