Nanoscale imaging of RNA with expansion microscopy

130

117

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

Section: Methodssupporting

confidence: 56%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Cahoon

Wang

et al. 2017

130

117

“…Reverse transcriptase may be rapidly distributed across a tissue for system-wide cDNA synthesis and subsequent in situ RNA sequencing, to extract transcriptomic data from the same intact tissue from which anatomical and physiological data were obtained (6). Rapid, diffusion-like dispersion of chemicals may also be useful for extending the application of emerging technologies [e.g., expansion microscopy (45)] to large intact tissues by enabling rapid transport of multifunctional probes into noncleared tissues. Finally, the concept of applying a rotational field to rapidly and evenly disperse particles throughout a tissue is not limited to electric force and may be generalizable to other types of forces, such as hydrodynamic pressure and magnetic force for molecular delivery.…”

Section: Discussionmentioning

confidence: 99%

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

Kim

Cho

Murray

et al. 2015

214

235

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1-3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.stochastic electrotransport | molecular transport | tissue clearing | tissue labeling | CLARITY

“…For this reason, a clearing method that preserves RNAs while removing proteins and lipids is desired for RNA FISH imaging. In the recently developed expansion microscopy method, proteins (25) and, more recently, RNAs (26,27) are physically anchored to a solvent-expandable and clearable poly-electrolyte matrix, effectively imprinting signals of these components on this matrix and allowing these molecular signals to be expanded along with the matrix for increasing image resolution. Inspired by this approach, we anchored RNA molecules to a nonswellable polyacrylamide (PA) matrix and then removed unwanted, non-RNA components, such as proteins and lipids, with the aim to remove their contribution to background fluorescence.…”

Section: Significancementioning

confidence: 99%

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Moffitt

Hao

Bambah-Mukku

et al. 2016

257

249

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, backgroundfrom off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.tissue clearing | fluorescence in situ hybridization | multiplexed imaging | single-cell transcriptomics | brain S ingle-molecule FISH (smFISH) is a powerful technique that allows the direct imaging of individual RNA molecules within single cells (1, 2). In this approach, RNAs are labeled via the hybridization of fluorescently labeled oligonucleotide probes, producing bright fluorescent spots for single RNA molecules, which reveal both the abundance and the spatial distribution of these RNAs inside cells (1, 2). The ability of smFISH to image gene expression at the single-cell level in both cell culture and tissue has led to exciting advances in our understanding of the natural noise in gene expression and its role in cellular response (3, 4), the intracellular spatial organization of RNAs and its role in posttranscriptional regulation (5, 6), and the spatial variation in gene expression within complex tissues and its role in the molecular definition of cell types and tissue functions (6, 7).To extend the benefits of this technique to systems-level questions and high-throughput gene-expression profiling, approaches to increase the multiplexing of smFISH (i.e., the number of different RNA species that can be simultaneously quantified within the same cell) have been developed (8-13). Most of these approaches take advantage of color multiplexing, which has allowed a few tens...