Navigating challenges in the application of superresolution microscopy

Lambert, Talley J.; Waters, Jennifer

doi:10.1083/jcb.201610011

Cited by 84 publications

(82 citation statements)

References 140 publications

(183 reference statements)

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“…For example, ExM SIM imaging with a biological (unexpanded) xy resolution of 100 nm, applied to a fourfoldexpanded sample, would effectively give a biological resolution of 25 nm, and, owing to the limits of optical physics, a biological resolution in the z dimension of 50-60 nm (25). However, a fourfold expansion presents a major challenge in performing conventional superresolution imaging, such as SIM (26). The distance between the expanded sample and the objective lens increases dramatically following expansion, making superresolution imaging nearly impossible.…”

Section: Resultsmentioning

confidence: 99%

Superresolution expansion microscopy reveals the three-dimensional organization of theDrosophilasynaptonemal complex

Cahoon

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

127

117

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The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.synaptonemal complex | expansion microscopy | meiosis | sister chromatids | structured illumination microscopy

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Section: Resultsmentioning

confidence: 99%

Superresolution expansion microscopy reveals the three-dimensional organization of theDrosophilasynaptonemal complex

Cahoon

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

127

117

View full text Add to dashboard Cite

show abstract

“…5C). The slightly larger R g obtained with IF can be explained by the sizes of primary and secondary antibodies that will place the fluorescent label at an offset distance from TRF1-bound telomeres and therefore lead to an apparent size increase (Lambert and Waters 2017).…”

Section: Telomeric Ddr In the Absence Of Decompactionmentioning

confidence: 99%

The telomeric DNA damage response occurs in the absence of chromatin decompaction

et al. 2017

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Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

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“…For instance, faster cameras with greater spatial resolution and higher sensitivity and brighter, more stable fluorophores would enable higher-quality images to be collected over longer periods of time than is currently possible. These advances would also help overcome some of the technical problems that currently limit most SR studies to fixed samples, namely, slow image acquisition rates, challenges in labelling proteins in living cells and phototoxicity associated with the high laser illumination intensities 113,114,118 . Thus, SR imaging might eventually be used to look at HR-related reaction mechanisms in living cells as they are occurring in real time.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

“…For instance, although SR studies of the synaptonemal complex have provided beautiful optical images of these structures and new insights into their organization, the impact of this organization on function has yet to be fully revealed 133–140 .While the potential of SR imaging is far from being fully realized, we anticipate continued and rapid developments in this powerful technology that will lead to broader applications 114,118 . For instance, traditional approaches have been unable to determine how chromosome organization contributes to homologue bias during meiotic recombination, but this problem may eventually prove amenable to SR imaging.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

A change of view: homologous recombination at single-molecule resolution

2017

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Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.

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Navigating challenges in the application of superresolution microscopy

Abstract: In this review, Lambert and Waters focus on the current practical limitations of superresolution microscopy (SRM) and provide information and resources to help biologists navigate through common pitfalls when designing an SRM experiment.

Cited by 84 publications

References 140 publications

Superresolution expansion microscopy reveals the three-dimensional organization of theDrosophilasynaptonemal complex

Superresolution expansion microscopy reveals the three-dimensional organization of theDrosophilasynaptonemal complex

The telomeric DNA damage response occurs in the absence of chromatin decompaction

A change of view: homologous recombination at single-molecule resolution

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