Multicolor Imaging: The Important Question of Co-Localization

Smallcombe, Anna

doi:10.2144/01306bt01

Cited by 43 publications

(38 citation statements)

References 1 publication

(1 reference statement)

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“…For visualization of light responsiveness of gene expression, imaging parameters were optimized for dark-grown samples, and all other samples were imaged at identical settings. Because high resolution is required to achieve visible separation of fluorescence signals that are in close proximity but are not spatially overlapping (Smallcombe, 2001), for colocalization analysis, leaves were observed with a 633 Planaprochromat oil (NA, 1.4; WD, 0.19 mm) objective of a Zeiss Axiovert 100M microscope equipped with a Zeiss LSM 510 laser module confocal unit (Carl Zeiss). For visualization of GFP and YFP, or of CFP and YFP, GFP or CFP was excited with the 458-nm line of an argon laser at 55% of output (equivalent to approximately 6 A) and 85% to 100% transmission and emission detected with a BP480-520 filter, while YFP was excited with the 514-nm line of an argon laser at 1% to 45% transmission and emission detected with a BP565-615 filter.…”

Section: Microtechniques and Microscopymentioning

confidence: 99%

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

et al. 2008

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Light provides crucial positional information in plant development, and the morphogenetic processes that are orchestrated by light signals are triggered by changes of gene expression in response to variations in light parameters. Control of expression of members of the RbcS and Lhc families of photosynthesis-associated nuclear genes by light cues is a paradigm for lightregulated gene transcription, but high-resolution expression profiles for these gene families are lacking. In this study, we have investigated expression patterns of members of the RbcS and Lhc gene families in Arabidopsis (Arabidopsis thaliana) at the cellular level during undisturbed development and upon controlled interference of the light environment. Members of the RbcS and Lhc gene families are expressed in specialized territories, including root tip, leaf adaxial, abaxial, and epidermal domains, and with distinct chronologies, identifying successive stages of leaf mesophyll ontogeny. Defined spatial and temporal overlap of gene expression fields suggest that the light-harvesting and photosynthetic apparatus may have a different polypeptide composition in different cells and that such composition could change over time even within the same cell.

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Section: Microtechniques and Microscopymentioning

confidence: 99%

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

et al. 2008

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“…The protocol assumes that images prepared for analysis were properly acquired and processed and are therefore suitable for quantification 3,7 . You should also ensure that samples were correctly prepared, that the microscope setup included the use of optimized emission filters and the proper pinhole size, and that images were acquired by sequential scanning and saved in original file format 8 .…”

Section: Experimental Designmentioning

confidence: 99%

“…Next, the proposed PPI 14 and a wellknown correlation coefficient 3,4 are used to estimate colocalization.…”

Section: Experimental Designmentioning

confidence: 99%

“…Visually, the overlap of the original colors of fluorophores will result in the appearance of a new, third, overlaid color, indicating colocalization. Observation of colocalization in fluorescence microscopy images was a significant advance for cell and molecular biological research 3,4 . Importantly, with the introduction of the ability to estimate the degree of this overlap quantitatively, it became possible to characterize it objectively as well [5][6][7] .…”

Section: Introductionmentioning

confidence: 99%

“…Importantly, with the introduction of the ability to estimate the degree of this overlap quantitatively, it became possible to characterize it objectively as well [5][6][7] . Previous work provided important information about the steps needed to perform quantification of colocalization properly, stressing the importance of: (i) avoiding the effect of bleed-through during acquisition of images; (ii) removing background fluorescence in them; and (iii) using optical sections rather than projections for its analysis 3,7,8 . Because of advances in digital imaging and a substantial increase in the use of fluorescence microscopy to perform spatial and temporal calculations in biology and medicine, deserved attention has been recently paid to the basic procedures of image acquisition, processing and handling to be followed to ensure accuracy of measurements in medico-biological images 9 .…”

Section: Introductionmentioning

confidence: 99%

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Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations

Zinchuk

Grossenbacher-Zinchuk

et al. 2011

Nat Protoc

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Interactions of proteins are examined by detecting their overlap using fluorescent markers. The observed overlap is then quantified to serve as a measure of spatial correlation. A major drawback of this approach is that it can produce false values because of the properties of the image background. To remedy this, we provide a protocol to reduce the contribution of image background and then apply a protein proximity index (PPI) and correlation coefficient to estimate colocalization. Background heterogeneity is reduced by the median filtering procedure, comprising two steps, to reduce random noise and background, respectively. Alternatively, background can be reduced by advanced thresholding. PPI provides separate values for each channel to characterize the contribution of each protein, whereas correlation coefficient determines the overall colocalization. The protocol is demonstrated using computer-simulated and real biological images. It minimizes human bias and can be universally applied to various cell types in which there is a need to understand protein-protein interactions. Background reductions require 3-5 min per image. Quantifications take <1 min. The entire procedure takes approximately 15-30 min.Published in " " which should be cited to refer to this work.

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Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

2008

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Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double‐stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of them for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations. Curr. Protoc. Cell Biol. 39:4.19.1‐4.19.16. © 2008 by John Wiley & Sons, Inc.

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Multicolor Imaging: The Important Question of Co-Localization

Cited by 43 publications

References 1 publication

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

Quantifying spatial correlations of fluorescent markers using enhanced background reduction with protein proximity index and correlation coefficient estimations

Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

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