Accuracy and precision in quantitative fluorescence microscopy

Waters, Jennifer

doi:10.1084/jem2067oia15

Cited by 124 publications

(157 citation statements)

References 60 publications

(125 reference statements)

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“…It allows for stable and strong fluorescence necessary to get SIM images of the appropriate quality with a high signal-to-noise ratio. In this approach, the top quality of the images is a must and, therefore, a careful control of all the steps necessary to gain the best quality images is a necessary prerequisite (Waters, 2009). Importantly, our method can reliably detect voids in DAPI staining as well as a drop in lamin B around NPCs.…”

Section: Discussionmentioning

confidence: 98%

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

Fišerová

Efenberková

Sieger

et al. 2017

Journal of Cell Science

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The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and nonactive chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatinmodifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.

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Section: Discussionmentioning

confidence: 98%

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

Fišerová

Efenberková

Sieger

et al. 2017

Journal of Cell Science

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“…Identical settings were used and images were taken within several days for each set of samples. A histogram of gray values was used to calculate relative GFP signal levels following methods for quantitative fluorescence microscopy (Waters 2009). The background signal for each image was determined using a region that did not contain worms and its average gray value per pixel and associated standard deviation were calculated using the Zeiss software.…”

Section: Construction Of Reportermentioning

confidence: 99%

Mutations in Caenorhabditis elegans eIF2β Permit Translation Initiation From Non-AUG Start Codons

Zhang

Maduzia

2010

Genetics

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Recognition of the AUG start codon on mRNAs during translation initiation in eukaryotes occurs in a preinitiation complex that includes small ribosomal subunits and multiple translation initiation factors. The complexity of this process and the lack of appropriate tools have prevented its genetic study in multicellular organisms. Here we describe a genetic system in the nematode Caenorhabditis elegans to study how the AUG start codon is selected. We have generated a sensitive reporter assay that allows for the isolation of mutants with reduced fidelity to recognize the AUG start codon. Two mutants were identified to have dominant missense mutations in iftb-1, which encodes the b-subunit of eIF2 (eIF2b). Both mutations occur in a conserved region located outside of the C2-C2 zinc finger domain where yeast SUI3 mutations are localized in Saccharomyces cerevisiae eIF2b. C. elegans iftb-1, as well as mutant eIF2bs carrying the equivalent SUI3 mutations, are able to initiate translation at non-AUG codons that retain two potential base-pairing interactions with the anticodon of the initiator methionyl tRNA. These analyses further support the critical role of eIF2b in start codon selection, and two functional domains within eIF2b are likely involved, one defined by our C. elegans mutants and the other by the yeast SUI3 mutants.

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“…As for the edge measurements we used the maximum value obtained. To get an accurate assessment of the change in immunostaining level in mutants it is essential to correct for nonspecific staining (Waters 2009). To do this we did similar measurements on clones of mwh 1 , a null allele.…”

Section: Methodsmentioning

confidence: 99%

The Drosophila Planar Polarity Proteins Inturned and Multiple Wing Hairs Interact Physically and Function Together

Yan²,

Adler

2010

Genetics

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The conserved frizzled ( fz) pathway regulates planar cell polarity in both vertebrate and invertebrate animals. This pathway has been most intensively studied in the wing of Drosophila, where the proteins encoded by pathway genes all accumulate asymmetrically. Upstream members of the pathway accumulate on the proximal, distal, or both cell edges in the vicinity of the adherens junction. More downstream components including Inturned and Multiple Wing Hairs accumulate on the proximal side of wing cells prior to hair initiation. The Mwh protein differs from other members of the pathway in also accumulating in growing hairs. Here we show that the two Mwh accumulation patterns are under different genetic control with the early proximal accumulation being regulated by the fz pathway and the latter hair accumulation being largely independent of the pathway. We also establish recruitment by proximally localized Inturned to be a putative mechanism for the localization of Mwh to the proximal side of wing cells. Genetically inturned (in) acts upstream of mwh (mwh) and is required for the proximal localization of Mwh. We show that Mwh can bind to and co-immunoprecipitate with Inturned. We also show that these two proteins can function in close juxtaposition in vivo. An InTMwh fusion protein provided complete rescue activity for both in and mwh mutations. The fusion protein localized to the proximal side of wing cells prior to hair formation and in growing hairs as expected if protein localization is a key for the function of these proteins.

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Accuracy and precision in quantitative fluorescence microscopy

Cited by 124 publications

References 60 publications

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

Mutations in Caenorhabditis elegans eIF2β Permit Translation Initiation From Non-AUG Start Codons

The Drosophila Planar Polarity Proteins Inturned and Multiple Wing Hairs Interact Physically and Function Together

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