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Optogenetic control of cell signaling pathway through scattering skull using wavefront shaping.

Yoon J, Lee M, Lee K, Kim N, Kim JM, Park J, Yu H, Choi C, Heo WD, Park Y - Sci Rep (2015)

Bottom Line: We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping.The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells.We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.

ABSTRACT
We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping. The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells. We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.

No MeSH data available.


Spatiotemporal regulation of intracellular Ca2+ level in HeLa cells co-expressing optoFGFR1 and R-GECO1, using wavefront shaping.(a) Targeted activation of Ca2+ signaling in HeLa cells after focusing through the skull. Gray color indicates R-GECO1 fluorescence signals. Green indicates excitation beam intensity through the skull without wavefront shaping, and cyan indicates optimized focus with wavefront shaping. The white arrow indicates the location of the optimized focus. The dashed lines indicate boundaries of individual cells. The red dashed line indicates the target cell. Quantitative analysis of R-GECO1 fluorescence signals obtained from cells i–iv in (a) with a plane wave (b) and with the shaped wave (c). The blue bar and the blue checked bar indicate irradiation time of a plane wave and the shaped wave, respectively. (d) Reversible control of Ca2+ level induced by optoFGFR1 in HeLa cells with repeated illumination using a shaped wave. Representative images show the baseline (left, no illumination) and maximal (right, shaped-wave illumination) R-GECO1 fluorescence signals. The dashed lines indicate each cell boundary. Red indicates the target cell. (e) Quantitative analysis of R-GECO1 fluorescence signals obtained from cells in (d). The cells were repeatedly illuminated using shaped waves. Each color matches the cells in (d). The blue-checked bars indicate irradiation time of the shaped wave.
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f3: Spatiotemporal regulation of intracellular Ca2+ level in HeLa cells co-expressing optoFGFR1 and R-GECO1, using wavefront shaping.(a) Targeted activation of Ca2+ signaling in HeLa cells after focusing through the skull. Gray color indicates R-GECO1 fluorescence signals. Green indicates excitation beam intensity through the skull without wavefront shaping, and cyan indicates optimized focus with wavefront shaping. The white arrow indicates the location of the optimized focus. The dashed lines indicate boundaries of individual cells. The red dashed line indicates the target cell. Quantitative analysis of R-GECO1 fluorescence signals obtained from cells i–iv in (a) with a plane wave (b) and with the shaped wave (c). The blue bar and the blue checked bar indicate irradiation time of a plane wave and the shaped wave, respectively. (d) Reversible control of Ca2+ level induced by optoFGFR1 in HeLa cells with repeated illumination using a shaped wave. Representative images show the baseline (left, no illumination) and maximal (right, shaped-wave illumination) R-GECO1 fluorescence signals. The dashed lines indicate each cell boundary. Red indicates the target cell. (e) Quantitative analysis of R-GECO1 fluorescence signals obtained from cells in (d). The cells were repeatedly illuminated using shaped waves. Each color matches the cells in (d). The blue-checked bars indicate irradiation time of the shaped wave.

Mentions: To test whether an optimized focus through the skull enables spatial control of light-sensitive proteins, we performed in vitro experiments using HeLa cells expressing optoFGFR1. OptoFGFR1 is a photoactivatable receptor tyrosine kinase which is activated by blue light (488 nm), and induces Ca2+ release from the endoplasmic reticulum through downstream signaling pathway16. To measure the intracellular Ca2+ level, R-GECO1, which emits red fluorescence by binding of Ca2+ ions, was co-expressed with optoFGFR1 in HeLa cells. First, the mouse skull was irradiated using an uncontrolled plane wave (6 μW) through a high-NA objective lens, for 60–200 s. The beam transmitted through the mouse skull exhibited a speckle pattern, and all the cells inside the field of view were irradiated by the speckle light field (Fig. 3a). After illumination with the plane wave, not only the target cell but also adjacent cells showed overall increases in R-GECO1 signals (Fig. 3b). Then, we optimized the wavefront of the incident beam in order to focus the beam in the middle of the target cell, 15 min after the plane wave illumination (Fig. 3a). Illumination with the optimized shaped wave, with less power (2 μW), specifically induced R-GECO1 signal increases only in the target cell; adjacent cells did not exhibit significant changes in R-GECO1 signals (Fig. 3c). These results demonstrate that the wavefront shaping method enables spatial control of light-sensitive proteins at the subcellular resolution to specifically activate individual single cells through the highly scattering skull layer.


Optogenetic control of cell signaling pathway through scattering skull using wavefront shaping.

Yoon J, Lee M, Lee K, Kim N, Kim JM, Park J, Yu H, Choi C, Heo WD, Park Y - Sci Rep (2015)

Spatiotemporal regulation of intracellular Ca2+ level in HeLa cells co-expressing optoFGFR1 and R-GECO1, using wavefront shaping.(a) Targeted activation of Ca2+ signaling in HeLa cells after focusing through the skull. Gray color indicates R-GECO1 fluorescence signals. Green indicates excitation beam intensity through the skull without wavefront shaping, and cyan indicates optimized focus with wavefront shaping. The white arrow indicates the location of the optimized focus. The dashed lines indicate boundaries of individual cells. The red dashed line indicates the target cell. Quantitative analysis of R-GECO1 fluorescence signals obtained from cells i–iv in (a) with a plane wave (b) and with the shaped wave (c). The blue bar and the blue checked bar indicate irradiation time of a plane wave and the shaped wave, respectively. (d) Reversible control of Ca2+ level induced by optoFGFR1 in HeLa cells with repeated illumination using a shaped wave. Representative images show the baseline (left, no illumination) and maximal (right, shaped-wave illumination) R-GECO1 fluorescence signals. The dashed lines indicate each cell boundary. Red indicates the target cell. (e) Quantitative analysis of R-GECO1 fluorescence signals obtained from cells in (d). The cells were repeatedly illuminated using shaped waves. Each color matches the cells in (d). The blue-checked bars indicate irradiation time of the shaped wave.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543936&req=5

f3: Spatiotemporal regulation of intracellular Ca2+ level in HeLa cells co-expressing optoFGFR1 and R-GECO1, using wavefront shaping.(a) Targeted activation of Ca2+ signaling in HeLa cells after focusing through the skull. Gray color indicates R-GECO1 fluorescence signals. Green indicates excitation beam intensity through the skull without wavefront shaping, and cyan indicates optimized focus with wavefront shaping. The white arrow indicates the location of the optimized focus. The dashed lines indicate boundaries of individual cells. The red dashed line indicates the target cell. Quantitative analysis of R-GECO1 fluorescence signals obtained from cells i–iv in (a) with a plane wave (b) and with the shaped wave (c). The blue bar and the blue checked bar indicate irradiation time of a plane wave and the shaped wave, respectively. (d) Reversible control of Ca2+ level induced by optoFGFR1 in HeLa cells with repeated illumination using a shaped wave. Representative images show the baseline (left, no illumination) and maximal (right, shaped-wave illumination) R-GECO1 fluorescence signals. The dashed lines indicate each cell boundary. Red indicates the target cell. (e) Quantitative analysis of R-GECO1 fluorescence signals obtained from cells in (d). The cells were repeatedly illuminated using shaped waves. Each color matches the cells in (d). The blue-checked bars indicate irradiation time of the shaped wave.
Mentions: To test whether an optimized focus through the skull enables spatial control of light-sensitive proteins, we performed in vitro experiments using HeLa cells expressing optoFGFR1. OptoFGFR1 is a photoactivatable receptor tyrosine kinase which is activated by blue light (488 nm), and induces Ca2+ release from the endoplasmic reticulum through downstream signaling pathway16. To measure the intracellular Ca2+ level, R-GECO1, which emits red fluorescence by binding of Ca2+ ions, was co-expressed with optoFGFR1 in HeLa cells. First, the mouse skull was irradiated using an uncontrolled plane wave (6 μW) through a high-NA objective lens, for 60–200 s. The beam transmitted through the mouse skull exhibited a speckle pattern, and all the cells inside the field of view were irradiated by the speckle light field (Fig. 3a). After illumination with the plane wave, not only the target cell but also adjacent cells showed overall increases in R-GECO1 signals (Fig. 3b). Then, we optimized the wavefront of the incident beam in order to focus the beam in the middle of the target cell, 15 min after the plane wave illumination (Fig. 3a). Illumination with the optimized shaped wave, with less power (2 μW), specifically induced R-GECO1 signal increases only in the target cell; adjacent cells did not exhibit significant changes in R-GECO1 signals (Fig. 3c). These results demonstrate that the wavefront shaping method enables spatial control of light-sensitive proteins at the subcellular resolution to specifically activate individual single cells through the highly scattering skull layer.

Bottom Line: We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping.The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells.We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.

ABSTRACT
We introduce a non-invasive approach for optogenetic regulation in biological cells through highly scattering skull tissue using wavefront shaping. The wavefront of the incident light was systematically controlled using a spatial light modulator in order to overcome multiple light-scattering in a mouse skull layer and to focus light on the target cells. We demonstrate that illumination with shaped waves enables spatiotemporal regulation of intracellular Ca(2+) level at the individual-cell level.

No MeSH data available.