Deconvolution and chromatic aberration corrections in quantifying colocalization of a transcription factor in three-dimensional cellular space

Thomas Abraham, Sarah E. Allan, Megan K. Levings

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

In the realm of multi-dimensional confocal microscopy, colocalization analysis of fluorescent emission signals has proven to be an invaluable tool for detecting molecular interactions between biological macromolecules at the subcellular level. We show here that image processing operations such as the deconvolution and chromatic corrections play a crucial role in the accurate determination of colocalization between biological macromolecules particularly when the fluorescent signals are faint, and when the fluorescent signals are in the blue and red emission regions. The cellular system presented here describes quantification of an activated forkhead box P3 (FOXP3) transcription factor in three-dimensional (3D) cellular space. 293T cells transfected with a conditionally active form of FOXP3 were stained for anti-FOXP3 conjugated to a florescent red dye (Phycoerythrin), and counterstained for DNA (nucleus) with fluorescent blue dye (Hoechst). Due to the broad emission spectra of these dyes, the fluorescent signals were collected only from peak regions and were acquired sequentially. Since the PE signal was weak, a confocal pinhole size of two Airy size was used to collect the 3D image data sets. The raw images supplemented with the spectral data show the preferential association of activated FOXP3 molecules with the nucleus. However, the PE signals were found to be highly diffusive and colocalization quantification from these raw images was not possible. In order to deconvolve the 3D raw image data set, point spread functions (PSFs) of these emissions were measured. From the measured PSF, we found that chromatic shifts between the blue and red colors were quite considerable. Followed by the applications of both the axial and lateral chromatic corrections, colocalization analysis performed on the deconvolved-chromatic corrected-3D image data set showed that 98% of DNA molecules were associated with FOXP3 molecules, whereas only 66% of FOXP3 molecules were colocalized with DNA molecules. In conclusion, our studies clearly demonstrate the importance of PSF measurements, chromatic aberration corrections followed by deconvolution in the accurate determination of transcription factors in the 3D cellular space. The reported imaging and processing methods can be a practical guide for quantitative fluorescence imaging of similar cellular systems and can provide a basis for further development.

Original languageEnglish (US)
Pages (from-to)633-640
Number of pages8
JournalMicron
Volume41
Issue number6
DOIs
StatePublished - Aug 1 2010

Fingerprint

Transcription Factors
Color
Fluorescent Dyes
DNA
Phycoerythrin
Forkhead Transcription Factors
HEK293 Cells
Optical Imaging
Confocal Microscopy
Coloring Agents
Datasets

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Cell Biology

Cite this

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title = "Deconvolution and chromatic aberration corrections in quantifying colocalization of a transcription factor in three-dimensional cellular space",
abstract = "In the realm of multi-dimensional confocal microscopy, colocalization analysis of fluorescent emission signals has proven to be an invaluable tool for detecting molecular interactions between biological macromolecules at the subcellular level. We show here that image processing operations such as the deconvolution and chromatic corrections play a crucial role in the accurate determination of colocalization between biological macromolecules particularly when the fluorescent signals are faint, and when the fluorescent signals are in the blue and red emission regions. The cellular system presented here describes quantification of an activated forkhead box P3 (FOXP3) transcription factor in three-dimensional (3D) cellular space. 293T cells transfected with a conditionally active form of FOXP3 were stained for anti-FOXP3 conjugated to a florescent red dye (Phycoerythrin), and counterstained for DNA (nucleus) with fluorescent blue dye (Hoechst). Due to the broad emission spectra of these dyes, the fluorescent signals were collected only from peak regions and were acquired sequentially. Since the PE signal was weak, a confocal pinhole size of two Airy size was used to collect the 3D image data sets. The raw images supplemented with the spectral data show the preferential association of activated FOXP3 molecules with the nucleus. However, the PE signals were found to be highly diffusive and colocalization quantification from these raw images was not possible. In order to deconvolve the 3D raw image data set, point spread functions (PSFs) of these emissions were measured. From the measured PSF, we found that chromatic shifts between the blue and red colors were quite considerable. Followed by the applications of both the axial and lateral chromatic corrections, colocalization analysis performed on the deconvolved-chromatic corrected-3D image data set showed that 98{\%} of DNA molecules were associated with FOXP3 molecules, whereas only 66{\%} of FOXP3 molecules were colocalized with DNA molecules. In conclusion, our studies clearly demonstrate the importance of PSF measurements, chromatic aberration corrections followed by deconvolution in the accurate determination of transcription factors in the 3D cellular space. The reported imaging and processing methods can be a practical guide for quantitative fluorescence imaging of similar cellular systems and can provide a basis for further development.",
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Deconvolution and chromatic aberration corrections in quantifying colocalization of a transcription factor in three-dimensional cellular space. / Abraham, Thomas; Allan, Sarah E.; Levings, Megan K.

In: Micron, Vol. 41, No. 6, 01.08.2010, p. 633-640.

Research output: Contribution to journalArticle

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N2 - In the realm of multi-dimensional confocal microscopy, colocalization analysis of fluorescent emission signals has proven to be an invaluable tool for detecting molecular interactions between biological macromolecules at the subcellular level. We show here that image processing operations such as the deconvolution and chromatic corrections play a crucial role in the accurate determination of colocalization between biological macromolecules particularly when the fluorescent signals are faint, and when the fluorescent signals are in the blue and red emission regions. The cellular system presented here describes quantification of an activated forkhead box P3 (FOXP3) transcription factor in three-dimensional (3D) cellular space. 293T cells transfected with a conditionally active form of FOXP3 were stained for anti-FOXP3 conjugated to a florescent red dye (Phycoerythrin), and counterstained for DNA (nucleus) with fluorescent blue dye (Hoechst). Due to the broad emission spectra of these dyes, the fluorescent signals were collected only from peak regions and were acquired sequentially. Since the PE signal was weak, a confocal pinhole size of two Airy size was used to collect the 3D image data sets. The raw images supplemented with the spectral data show the preferential association of activated FOXP3 molecules with the nucleus. However, the PE signals were found to be highly diffusive and colocalization quantification from these raw images was not possible. In order to deconvolve the 3D raw image data set, point spread functions (PSFs) of these emissions were measured. From the measured PSF, we found that chromatic shifts between the blue and red colors were quite considerable. Followed by the applications of both the axial and lateral chromatic corrections, colocalization analysis performed on the deconvolved-chromatic corrected-3D image data set showed that 98% of DNA molecules were associated with FOXP3 molecules, whereas only 66% of FOXP3 molecules were colocalized with DNA molecules. In conclusion, our studies clearly demonstrate the importance of PSF measurements, chromatic aberration corrections followed by deconvolution in the accurate determination of transcription factors in the 3D cellular space. The reported imaging and processing methods can be a practical guide for quantitative fluorescence imaging of similar cellular systems and can provide a basis for further development.

AB - In the realm of multi-dimensional confocal microscopy, colocalization analysis of fluorescent emission signals has proven to be an invaluable tool for detecting molecular interactions between biological macromolecules at the subcellular level. We show here that image processing operations such as the deconvolution and chromatic corrections play a crucial role in the accurate determination of colocalization between biological macromolecules particularly when the fluorescent signals are faint, and when the fluorescent signals are in the blue and red emission regions. The cellular system presented here describes quantification of an activated forkhead box P3 (FOXP3) transcription factor in three-dimensional (3D) cellular space. 293T cells transfected with a conditionally active form of FOXP3 were stained for anti-FOXP3 conjugated to a florescent red dye (Phycoerythrin), and counterstained for DNA (nucleus) with fluorescent blue dye (Hoechst). Due to the broad emission spectra of these dyes, the fluorescent signals were collected only from peak regions and were acquired sequentially. Since the PE signal was weak, a confocal pinhole size of two Airy size was used to collect the 3D image data sets. The raw images supplemented with the spectral data show the preferential association of activated FOXP3 molecules with the nucleus. However, the PE signals were found to be highly diffusive and colocalization quantification from these raw images was not possible. In order to deconvolve the 3D raw image data set, point spread functions (PSFs) of these emissions were measured. From the measured PSF, we found that chromatic shifts between the blue and red colors were quite considerable. Followed by the applications of both the axial and lateral chromatic corrections, colocalization analysis performed on the deconvolved-chromatic corrected-3D image data set showed that 98% of DNA molecules were associated with FOXP3 molecules, whereas only 66% of FOXP3 molecules were colocalized with DNA molecules. In conclusion, our studies clearly demonstrate the importance of PSF measurements, chromatic aberration corrections followed by deconvolution in the accurate determination of transcription factors in the 3D cellular space. The reported imaging and processing methods can be a practical guide for quantitative fluorescence imaging of similar cellular systems and can provide a basis for further development.

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