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Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta.

Kodama Y - PLoS ONE (2016)

Bottom Line: Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants.In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence.As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.

View Article: PubMed Central - PubMed

Affiliation: Center for Bioscience Research and Education, Utsunomiya University, Tochigi, 321-8505, Japan.

ABSTRACT
Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.

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Fluorescence imaging of Mpphot-Citrine on the chloroplast periphery of transgenic Marchantia polymorpha.(a) Single cellular fluorescence imaging. (b) Intracellular fluorescence imaging. Upper and lower panels reveal gate-off and gate-on (time: 0.3–12.0 ns) images, respectively. Left and center panels show images at the yellow (520–561 nm) and red (648–709 nm) wavelength regions, respectively. The right panels demonstrate merged images comprising yellow and red images. Scale bar, 10 μm.
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pone.0152484.g002: Fluorescence imaging of Mpphot-Citrine on the chloroplast periphery of transgenic Marchantia polymorpha.(a) Single cellular fluorescence imaging. (b) Intracellular fluorescence imaging. Upper and lower panels reveal gate-off and gate-on (time: 0.3–12.0 ns) images, respectively. Left and center panels show images at the yellow (520–561 nm) and red (648–709 nm) wavelength regions, respectively. The right panels demonstrate merged images comprising yellow and red images. Scale bar, 10 μm.

Mentions: To demonstrate the practical use of the time-gated rejection of chloroplast autofluorescence, the fluorescent signal around the chloroplast was visualized. As a model experiment, an improved YFP (Citrine)-tagged phototropin (phot), a blue-light photoreceptor localized at the chloroplast outer envelope membrane, was analyzed in M. polymorpha. Note that the lifetime of Citrine fluorescence is approximately 3.6 ns [13], and only 10% of Citrine fluorescence was reduced by the time gating with 0.3–12.0 ns (S3 Fig). Previous studies on A. thaliana reported that phot localizes on the plasma membrane in darkness, whereas some fractions translocate into the cytosol or Golgi apparatus in response to blue light [14,15]. Recently, it was also reported that phot1 and phot2 localize on the chloroplast outer envelope membrane in A. thaliana [16]. However, in other plant species, the presence of phot on the chloroplast outer envelope membrane remains to be determined. In M. polymorpha, phot is encoded by a single copy gene, which is termed “Mpphot” [17]. A previous study reported only the localization of Mpphot-Citrine at the plasma membrane [17]. In the current study, transgenic liverwort expressing Mpphot-Citrine was produced by the AgarTrap method, a genetic transformation method for M. polymorpha [11]. Confocal laser microscopic analysis determined that Mpphot-Citrine localizes not only on the plasma membrane but also on the chloroplast periphery (Fig 2A, upper panels). Using the time-gated fluorescence imaging technique with a 514-nm laser, the Citrine fluorescent signal at the chloroplast periphery was clearly visualized, with no chloroplast autofluorescence at a yellow wavelength region (520–561 nm) (Fig 2A, lower panels). When chloroplasts were scanned with a zoom, the autofluorescence at the yellow wavelength region was significantly increased, highly compromising the interpretation of the image data (Fig 2B, upper panels). When the time-gated technique was used, the autofluorescence was completely eliminated, thereby allowing a clearer visualization of Citrine fluorescence at the chloroplast periphery (Fig 2B, lower panels). The observations suggested the presence of Mpphot-Citrine on the chloroplast outer envelope membrane of M. polymorpha.


Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta.

Kodama Y - PLoS ONE (2016)

Fluorescence imaging of Mpphot-Citrine on the chloroplast periphery of transgenic Marchantia polymorpha.(a) Single cellular fluorescence imaging. (b) Intracellular fluorescence imaging. Upper and lower panels reveal gate-off and gate-on (time: 0.3–12.0 ns) images, respectively. Left and center panels show images at the yellow (520–561 nm) and red (648–709 nm) wavelength regions, respectively. The right panels demonstrate merged images comprising yellow and red images. Scale bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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pone.0152484.g002: Fluorescence imaging of Mpphot-Citrine on the chloroplast periphery of transgenic Marchantia polymorpha.(a) Single cellular fluorescence imaging. (b) Intracellular fluorescence imaging. Upper and lower panels reveal gate-off and gate-on (time: 0.3–12.0 ns) images, respectively. Left and center panels show images at the yellow (520–561 nm) and red (648–709 nm) wavelength regions, respectively. The right panels demonstrate merged images comprising yellow and red images. Scale bar, 10 μm.
Mentions: To demonstrate the practical use of the time-gated rejection of chloroplast autofluorescence, the fluorescent signal around the chloroplast was visualized. As a model experiment, an improved YFP (Citrine)-tagged phototropin (phot), a blue-light photoreceptor localized at the chloroplast outer envelope membrane, was analyzed in M. polymorpha. Note that the lifetime of Citrine fluorescence is approximately 3.6 ns [13], and only 10% of Citrine fluorescence was reduced by the time gating with 0.3–12.0 ns (S3 Fig). Previous studies on A. thaliana reported that phot localizes on the plasma membrane in darkness, whereas some fractions translocate into the cytosol or Golgi apparatus in response to blue light [14,15]. Recently, it was also reported that phot1 and phot2 localize on the chloroplast outer envelope membrane in A. thaliana [16]. However, in other plant species, the presence of phot on the chloroplast outer envelope membrane remains to be determined. In M. polymorpha, phot is encoded by a single copy gene, which is termed “Mpphot” [17]. A previous study reported only the localization of Mpphot-Citrine at the plasma membrane [17]. In the current study, transgenic liverwort expressing Mpphot-Citrine was produced by the AgarTrap method, a genetic transformation method for M. polymorpha [11]. Confocal laser microscopic analysis determined that Mpphot-Citrine localizes not only on the plasma membrane but also on the chloroplast periphery (Fig 2A, upper panels). Using the time-gated fluorescence imaging technique with a 514-nm laser, the Citrine fluorescent signal at the chloroplast periphery was clearly visualized, with no chloroplast autofluorescence at a yellow wavelength region (520–561 nm) (Fig 2A, lower panels). When chloroplasts were scanned with a zoom, the autofluorescence at the yellow wavelength region was significantly increased, highly compromising the interpretation of the image data (Fig 2B, upper panels). When the time-gated technique was used, the autofluorescence was completely eliminated, thereby allowing a clearer visualization of Citrine fluorescence at the chloroplast periphery (Fig 2B, lower panels). The observations suggested the presence of Mpphot-Citrine on the chloroplast outer envelope membrane of M. polymorpha.

Bottom Line: Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants.In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence.As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.

View Article: PubMed Central - PubMed

Affiliation: Center for Bioscience Research and Education, Utsunomiya University, Tochigi, 321-8505, Japan.

ABSTRACT
Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.

Show MeSH