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Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus

Photoconversion of purified EGFP in vitro.(a) Typical pre- and post-photoconversion images of a thin (~10 μm) layer of 30 μM EGFP using 405 nm excitation delivered through a 1.2 NA objective to the region within the circle using the Zen software bleaching mode (Green channel: 505–525 nm; Red channel: 580–670 nm; scale bar: 50 μm). See also Supplementary Movie S1. 10 iterations of 257 ms each were performed. (b) Emission spectra of photoconverted red state EGFP acquired using the Zeiss 710 spectral detector (5 nm bandwidth intervals). (c) Absorption spectra of EGFP (~2 μM) at pH 5.0 and pH 8.0. (d) Time course of the increase in red fluorescence with successive 405 nm irradiations within the region of interest (ROI). EGFP (30 mM) was subjected to a sequence of 10 “bleach” iterations over the ROI (1.8 seconds) followed by image acquisition using 488 and 561 nm illumination. The 405 nm power at the sample was at 2.5 mW, corresponding to 6.8 MW/cm2 at the focus of the 1.2 NA objective lens. Plotted data points are the average pixel values across the entire field of view which estimates the total photo-product produced. Error bars represent the SEM of three trials at pH 5.0 and two at pH 8.0. Black lines are fits to exponential increase for the red channel (1-exp(-αt)) and decrease (exp(-αt)) for the green. Returned values of α were ~0.14 s−1 for all data sets except the decay of the pH 8.0 green channel (which was 0.24 s−1).
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f2: Photoconversion of purified EGFP in vitro.(a) Typical pre- and post-photoconversion images of a thin (~10 μm) layer of 30 μM EGFP using 405 nm excitation delivered through a 1.2 NA objective to the region within the circle using the Zen software bleaching mode (Green channel: 505–525 nm; Red channel: 580–670 nm; scale bar: 50 μm). See also Supplementary Movie S1. 10 iterations of 257 ms each were performed. (b) Emission spectra of photoconverted red state EGFP acquired using the Zeiss 710 spectral detector (5 nm bandwidth intervals). (c) Absorption spectra of EGFP (~2 μM) at pH 5.0 and pH 8.0. (d) Time course of the increase in red fluorescence with successive 405 nm irradiations within the region of interest (ROI). EGFP (30 mM) was subjected to a sequence of 10 “bleach” iterations over the ROI (1.8 seconds) followed by image acquisition using 488 and 561 nm illumination. The 405 nm power at the sample was at 2.5 mW, corresponding to 6.8 MW/cm2 at the focus of the 1.2 NA objective lens. Plotted data points are the average pixel values across the entire field of view which estimates the total photo-product produced. Error bars represent the SEM of three trials at pH 5.0 and two at pH 8.0. Black lines are fits to exponential increase for the red channel (1-exp(-αt)) and decrease (exp(-αt)) for the green. Returned values of α were ~0.14 s−1 for all data sets except the decay of the pH 8.0 green channel (which was 0.24 s−1).

Mentions: To verify whether EGFP could be photoconverted in vitro from its normal green state to a red state, we investigated irradiation of purified EGFP. By using the Zeiss 710 bleaching mode with a 405 nm laser for photoconversion, rather than 488 nm as used in the previous reports, purified EGFP was imaged before and after application of the 405 nm light. The green-state of purified EGFP was imaged with excitation at 488 nm, and the emission was acquired between 505–525 nm. The photoconverted EGFP (red state) was monitored using 561 nm excitation and detected between 580–670 nm. Photoconversion occurred, as demonstrated by the decrease in green fluorescence and the simultaneous increase in red fluorescence (Fig. 2a). We observed a maximum emission peak around 612 nm when using 561 nm excitation (Fig. 2b). The red species of EGFP is not excited by 488 nm light and we could detect no red species formed using 488 nm light alone for photoconversion.


Green to red photoconversion of GFP for protein tracking in vivo.

Sattarzadeh A, Saberianfar R, Zipfel WR, Menassa R, Hanson MR - Sci Rep (2015)

Photoconversion of purified EGFP in vitro.(a) Typical pre- and post-photoconversion images of a thin (~10 μm) layer of 30 μM EGFP using 405 nm excitation delivered through a 1.2 NA objective to the region within the circle using the Zen software bleaching mode (Green channel: 505–525 nm; Red channel: 580–670 nm; scale bar: 50 μm). See also Supplementary Movie S1. 10 iterations of 257 ms each were performed. (b) Emission spectra of photoconverted red state EGFP acquired using the Zeiss 710 spectral detector (5 nm bandwidth intervals). (c) Absorption spectra of EGFP (~2 μM) at pH 5.0 and pH 8.0. (d) Time course of the increase in red fluorescence with successive 405 nm irradiations within the region of interest (ROI). EGFP (30 mM) was subjected to a sequence of 10 “bleach” iterations over the ROI (1.8 seconds) followed by image acquisition using 488 and 561 nm illumination. The 405 nm power at the sample was at 2.5 mW, corresponding to 6.8 MW/cm2 at the focus of the 1.2 NA objective lens. Plotted data points are the average pixel values across the entire field of view which estimates the total photo-product produced. Error bars represent the SEM of three trials at pH 5.0 and two at pH 8.0. Black lines are fits to exponential increase for the red channel (1-exp(-αt)) and decrease (exp(-αt)) for the green. Returned values of α were ~0.14 s−1 for all data sets except the decay of the pH 8.0 green channel (which was 0.24 s−1).
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Related In: Results  -  Collection

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f2: Photoconversion of purified EGFP in vitro.(a) Typical pre- and post-photoconversion images of a thin (~10 μm) layer of 30 μM EGFP using 405 nm excitation delivered through a 1.2 NA objective to the region within the circle using the Zen software bleaching mode (Green channel: 505–525 nm; Red channel: 580–670 nm; scale bar: 50 μm). See also Supplementary Movie S1. 10 iterations of 257 ms each were performed. (b) Emission spectra of photoconverted red state EGFP acquired using the Zeiss 710 spectral detector (5 nm bandwidth intervals). (c) Absorption spectra of EGFP (~2 μM) at pH 5.0 and pH 8.0. (d) Time course of the increase in red fluorescence with successive 405 nm irradiations within the region of interest (ROI). EGFP (30 mM) was subjected to a sequence of 10 “bleach” iterations over the ROI (1.8 seconds) followed by image acquisition using 488 and 561 nm illumination. The 405 nm power at the sample was at 2.5 mW, corresponding to 6.8 MW/cm2 at the focus of the 1.2 NA objective lens. Plotted data points are the average pixel values across the entire field of view which estimates the total photo-product produced. Error bars represent the SEM of three trials at pH 5.0 and two at pH 8.0. Black lines are fits to exponential increase for the red channel (1-exp(-αt)) and decrease (exp(-αt)) for the green. Returned values of α were ~0.14 s−1 for all data sets except the decay of the pH 8.0 green channel (which was 0.24 s−1).
Mentions: To verify whether EGFP could be photoconverted in vitro from its normal green state to a red state, we investigated irradiation of purified EGFP. By using the Zeiss 710 bleaching mode with a 405 nm laser for photoconversion, rather than 488 nm as used in the previous reports, purified EGFP was imaged before and after application of the 405 nm light. The green-state of purified EGFP was imaged with excitation at 488 nm, and the emission was acquired between 505–525 nm. The photoconverted EGFP (red state) was monitored using 561 nm excitation and detected between 580–670 nm. Photoconversion occurred, as demonstrated by the decrease in green fluorescence and the simultaneous increase in red fluorescence (Fig. 2a). We observed a maximum emission peak around 612 nm when using 561 nm excitation (Fig. 2b). The red species of EGFP is not excited by 488 nm light and we could detect no red species formed using 488 nm light alone for photoconversion.

Bottom Line: A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms.We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cornell University, Department of Molecular Biology and Genetics, Ithaca, NY, 14853 USA.

ABSTRACT
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.

No MeSH data available.


Related in: MedlinePlus