Extending resolution within a single imaging frame

Torres, Esley; Pinto‐Cámara, Raúl; Linares, Alejandro; Martínez, Damián; Abonza, Víctor; Brito‐Alarcón, Eduardo; Cruz, C.; Valdés-Galindo, Gustavo; Torres, David; Jabloñski, Martina; Torres-Martínez, Héctor H.; Martínez, José Luis; Hernández, Haydee O.; Ocelotl-Oviedo, Jose Pablo; Suárez, Yasel Garcés; Barchi, Marco; D’Antuono, Rocco; Bošković, Ana; Dubrovsky, Joseph G.; Darszon, Alberto; Buffone, Mariano G.; Morales, Roberto; Rendón-Mancha, Juan Manuel; Wood, Christopher D.; Hernández-García, Armando; Krapf, Diego; Crevenna, Álvaro H.; Guerrero, Adán

doi:10.1038/s41467-022-34693-9

Cited by 35 publications

(24 citation statements)

References 95 publications

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“…In the negative control (no acrosomal exocytosis), the diameter remained unchanged. This phenomenon was also observed using mean shift super-resolution (MSSR) (18). To further support this observation, other plasma membrane probes of different molecular structure, such as Memglow 700 (Figure S2A), Bodipy-GM1 (Figure S2B), and FM1-43 (Figure S2C), were used.…”

Section: Supplementary Movie S3)supporting

confidence: 60%

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Jabloñski

Luque

Gómez-Elías

et al. 2023

Preprint

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Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. Within the midpiece of the sperm flagellum lies a cortical network of actin that is arranged as a double helix sheltering the mitochondrial sheath. This work demonstrates that the actin network of the midpiece undergoes structural changes that result in motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo acrosomal exocytosis, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation during the fusion process. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization.

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Section: Supplementary Movie S3)supporting

confidence: 60%

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Jabloñski

Luque

Gómez-Elías

et al. 2023

Preprint

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“…The initial implementation of SRRF resulted in a 50-70 nm resolution 6 , leading to the impression that this is the best achievable resolution for this technique, as it is implied by some subsequent works (e.g. 32 ). This is not the case, as demonstrated in our work on nanorulers ( Supplementary Figs.…”

Section: Supplementary Discussionmentioning

confidence: 93%

“…100-300 frames), thus rendering it a method of choice for live-cell SRRF 6, 27 . TRA is heavily dependent on the distance between the fluorophores, and performs best when the different fluorescent objects are separated by more than 70% of the full width at half maximum (FWHM) of the point-spread-function (PSF 32 ). This implies that this procedure is not intended to produce a very high resolution, unlike the TRAC analyses.…”

Section: Supplementary Discussionmentioning

confidence: 99%

“…This, however, will result in a lower signal-to-noise ratio (SNR), which is an essential parameter for all fluctuation-based analyses. Even when applied to low numbers of frames, SRRF provided excellent images when the signal-to-noise ratio surpassed 10-15 6, 32 . Below these values, SRRF will perform more poorly than many other related methods, as MUSICAL or ESI 32 , implying that users should consider carefully the noise levels of their images, as explained in Supplementary Fig.…”

Section: Supplementary Discussionmentioning

confidence: 99%

“…Working with low frame numbers reduces the achievable resolution, even when working with ExM gels 20, 31 . Second, the signal-to-noise ratio needs to be optimized carefully 32 , as we also demonstrate in Supplementary Fig. 4 .…”

Section: Introductionmentioning

confidence: 99%

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Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

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Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

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The plant early recombinosome: a high security complex to break DNA during meiosis

Rafiei,

Ronceret

2024

Plant Reprod

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Key message The formacion of numerous unpredictable DNA Double Strand Breaks (DSBs) on chromosomes iniciates meiotic recombination. In this perspective, we propose a ‘multi-key lock’ model to secure the risky but necesary breaks as well as a ‘one per pair of cromatids’ model for the topoisomerase-like early recombinosome. Abstract During meiosis, homologous chromosomes recombine at few sites of crossing-overs (COs) to ensure correct segregation. The initiation of meiotic recombination involves the formation of DNA double strand breaks (DSBs) during prophase I. Too many DSBs are dangerous for genome integrity: if these DSBs are not properly repaired, it could potentially lead to chromosomal fragmentation. Too few DSBs are also problematic: if the obligate CO cannot form between bivalents, catastrophic unequal segregation of univalents lead to the formation of sterile aneuploid spores. Research on the regulation of the formation of these necessary but risky DSBs has recently advanced in yeast, mammals and plants. DNA DSBs are created by the enzymatic activity of the early recombinosome, a topoisomerase-like complex containing SPO11. This opinion paper reviews recent insights on the regulation of the SPO11 cofactors necessary for the introduction of temporally and spatially controlled DSBs. We propose that a ‘multi-key-lock’ model for each subunit of the early recombinosome complex is required to secure the formation of DSBs. We also discuss the hypothetical implications that the established topoisomerase-like nature of the SPO11 core-complex can have in creating DSB in only one of the two replicated chromatids of early prophase I meiotic chromosomes. This hypothetical ‘one per pair of chromatids’ DSB formation model could optimize the faithful repair of the self-inflicted DSBs. Each DSB could use three potential intact homologous DNA sequences as repair template: one from the sister chromatid and the two others from the homologous chromosomes.

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Extending resolution within a single imaging frame

Cited by 35 publications

References 95 publications

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Visualizing proteins by expansion microscopy

The plant early recombinosome: a high security complex to break DNA during meiosis

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