7 research outputs found
Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs
In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage
Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs
In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage
Highly Activatable and Environment-Insensitive Optical Highlighters for Selective Spatiotemporal Imaging of Target Proteins
Optical highlighters are photoactivatable fluorescent
molecules
that exhibit pronounced changes in their spectral properties in response
to irradiation with light of a specific wavelength and intensity.
Here, we present a novel design strategy for a new class of caged
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-<i>s</i>-indacene)
fluorophores, based on the use of photoremovable protecting groups
(PRPGs) with high reduction potentials that serve as both a photosensitive
unit and a fluorescence quencher via photoinduced electron transfer
(PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated,
with activation ratios exceeding 600-fold in aqueous solutions. We
then combined this photoactivatable fluorophore with a SNAP (mutant
of <i>O</i><sup>6</sup>-alkylguanine DNA alkyltransferase)
ligand to obtain a small-molecule-based optical highlighter for visualization
of protein dynamics, using the well-established SNAP tag technology.
As proof of concept, we demonstrate spatiotemporal imaging of the
fusion protein of epidermal growth factor receptor (EGFR) with SNAP
tag in living cells. We also demonstrate highlighting of cells of
interest in live zebrafish embryos, using the fusion protein of histone
2A with SNAP tag
Highly Activatable and Environment-Insensitive Optical Highlighters for Selective Spatiotemporal Imaging of Target Proteins
Optical highlighters are photoactivatable fluorescent
molecules
that exhibit pronounced changes in their spectral properties in response
to irradiation with light of a specific wavelength and intensity.
Here, we present a novel design strategy for a new class of caged
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-<i>s</i>-indacene)
fluorophores, based on the use of photoremovable protecting groups
(PRPGs) with high reduction potentials that serve as both a photosensitive
unit and a fluorescence quencher via photoinduced electron transfer
(PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated,
with activation ratios exceeding 600-fold in aqueous solutions. We
then combined this photoactivatable fluorophore with a SNAP (mutant
of <i>O</i><sup>6</sup>-alkylguanine DNA alkyltransferase)
ligand to obtain a small-molecule-based optical highlighter for visualization
of protein dynamics, using the well-established SNAP tag technology.
As proof of concept, we demonstrate spatiotemporal imaging of the
fusion protein of epidermal growth factor receptor (EGFR) with SNAP
tag in living cells. We also demonstrate highlighting of cells of
interest in live zebrafish embryos, using the fusion protein of histone
2A with SNAP tag
Highly Activatable and Environment-Insensitive Optical Highlighters for Selective Spatiotemporal Imaging of Target Proteins
Optical highlighters are photoactivatable fluorescent
molecules
that exhibit pronounced changes in their spectral properties in response
to irradiation with light of a specific wavelength and intensity.
Here, we present a novel design strategy for a new class of caged
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-<i>s</i>-indacene)
fluorophores, based on the use of photoremovable protecting groups
(PRPGs) with high reduction potentials that serve as both a photosensitive
unit and a fluorescence quencher via photoinduced electron transfer
(PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated,
with activation ratios exceeding 600-fold in aqueous solutions. We
then combined this photoactivatable fluorophore with a SNAP (mutant
of <i>O</i><sup>6</sup>-alkylguanine DNA alkyltransferase)
ligand to obtain a small-molecule-based optical highlighter for visualization
of protein dynamics, using the well-established SNAP tag technology.
As proof of concept, we demonstrate spatiotemporal imaging of the
fusion protein of epidermal growth factor receptor (EGFR) with SNAP
tag in living cells. We also demonstrate highlighting of cells of
interest in live zebrafish embryos, using the fusion protein of histone
2A with SNAP tag
Highly Activatable and Environment-Insensitive Optical Highlighters for Selective Spatiotemporal Imaging of Target Proteins
Optical highlighters are photoactivatable fluorescent
molecules
that exhibit pronounced changes in their spectral properties in response
to irradiation with light of a specific wavelength and intensity.
Here, we present a novel design strategy for a new class of caged
BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-<i>s</i>-indacene)
fluorophores, based on the use of photoremovable protecting groups
(PRPGs) with high reduction potentials that serve as both a photosensitive
unit and a fluorescence quencher via photoinduced electron transfer
(PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated,
with activation ratios exceeding 600-fold in aqueous solutions. We
then combined this photoactivatable fluorophore with a SNAP (mutant
of <i>O</i><sup>6</sup>-alkylguanine DNA alkyltransferase)
ligand to obtain a small-molecule-based optical highlighter for visualization
of protein dynamics, using the well-established SNAP tag technology.
As proof of concept, we demonstrate spatiotemporal imaging of the
fusion protein of epidermal growth factor receptor (EGFR) with SNAP
tag in living cells. We also demonstrate highlighting of cells of
interest in live zebrafish embryos, using the fusion protein of histone
2A with SNAP tag