FISH-quant: automatic counting of transcripts in 3D FISH images

Mueller, Florian; Senecal, Adrien; Tantale, Katjana; Marie-Nelly, Hervé; Ly, Nathalie; Collin, Olivier; Basyuk, Eugénia; Bertrand, Édouard; Darzacq, Xavier; Zimmer, Christophe

doi:10.1038/nmeth.2406

Cited by 360 publications

(399 citation statements)

References 6 publications

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“…To detect transcripts, we filtered images, detected local maxima of intensity and used a threshold to separate true transcripts from background noise, similar to previous approaches (Lyubimova et al, 2013;Mueller et al, 2013;Raj et al, 2008). To determine the appropriate threshold for detection of ntla transcripts, we plotted the intensity distribution of all detected maxima (Fig.…”

Section: Results and Discussion Sensitive And Specific Detection And mentioning

confidence: 99%

“…Furthermore, neither technique provides subcellular resolution. The development of single-molecule fluorescence in situ hybridization (smFISH) has enabled the detection of individual transcripts both in single cells and tissues (Bahar Halpern et al, 2015;Battich et al, 2013;Boettiger and Levine, 2013;Itzkovitz et al, 2012Itzkovitz et al, , 2011Little et al, 2013;Lyubimova et al, 2013;Mueller et al, 2013;Nair et al, 2013;Oka and Sato, 2015;Peterson et al, 2012;Raj et al, 2008). This technical advance has, for example, improved our understanding of the design principles of the developing mouse intestine (Itzkovitz et al, 2012) and the establishment of precise developmental gene expression patterns in Drosophila blastoderm embryos (Boettiger and Levine, 2013;Little et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

et al. 2015

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Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.

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Section: Results and Discussion Sensitive And Specific Detection And mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

et al. 2015

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“…(A) Histogram depicting the distribution of the number of mRNA granules/cell for each strain. (B) Graph depicting the estimated number of MFA2 mRNA transcripts per mRNA granule, as calculated using the transcription-site analysis tool of the FISH-quant program (Mueller et al 2013). Each symbol on the graphrepresents a single mRNA granule.…”

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confidence: 99%

Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to “MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system”

Haimovich

Zabezhinsky

Haas

et al. 2016

RNA

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The MS2 system has been extensively used to visualize single mRNA molecules in live cells and follow their localization and behavior. In their Letter to the Editor recently published, Garcia and Parker suggest that use of the MS2 system may yield erroneous mRNA localization results due to the accumulation of 3 ′ decay products. Here we cite published works and provide new data which demonstrate that this is not a phenomenon general to endogenously expressed MS2-tagged transcripts, and that some of the results obtained in their study could have arisen from artifacts of gene expression.

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“…Second, the image panels are split and aligned using the transformation matrix generated in the bead calibration step. Third, the relative intensities of the different panels are corrected; 3D Gaussian fitting of individual 3D PSFs was performed with the freely available FishQUANT software (35) to retrieve the emitter position, and 3D superresolution images were visualized with ViSP software (29).…”

Section: Methodsmentioning

confidence: 99%

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

Hajj

Wiśniewski

Beheiry

et al. 2014

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

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Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 μm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-μm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.superresolution | 3D localization | microscopy | multiplane imaging | single-molecule fluorescence

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FISH-quant: automatic counting of transcripts in 3D FISH images

Cited by 360 publications

References 6 publications

Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to “MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system”

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

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