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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.


Related in: MedlinePlus

Optical focusing through a skull layer using wavefront shaping technique.(a) Schematic of the experiment. Cells expressing both optoFGFR1 and R-GECO1 are placed above the skull layer. When illuminating with a plane wave through an objective lens, the beam undergoes multiple light scattering as it propagates through the skull and this diffused light may activate optigenetic signals in all cells in an uncontrolled manner (left). By shaping the wavefront of the impinging beam using SLM, optical focus can be generated at the plane of the cells, thus activating optogenetic regulation at the level of individual cells (right). (b) Experimental setup, L1–3 lens: The blue dashed box indicates a wavefront shaping part, and the red dashed box indicates a fluorescence-imaging part. (c) Confocal image of second harmonic generation signals in the used mouse skull on the x-y plane (left) and in the axial direction (right). Scale bar 300 μm. (d) Intensity images of the transmitted beam through the skull without wavefront shaping (left), and with the wavefront shaping (right). (e) Measured intensity enhancements according to segment numbers and samples. (f) Measured intensity of optimized foci (n = 5) and intensity enhancements (n = 5) according to numbers of skull layers. The graph inset illustrates the optical transmittance according to numbers of skull layers. The circles represent experimental data, and a dashed line is a theoretically expected from Eq. (1). (g) Measured transport mean free path of the frontal (n = 4) and parietal bones (n = 4), respectively. Schematic of a mouse skull (top) and corresponding transport mean free paths of the mouse skull (bottom).
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f2: Optical focusing through a skull layer using wavefront shaping technique.(a) Schematic of the experiment. Cells expressing both optoFGFR1 and R-GECO1 are placed above the skull layer. When illuminating with a plane wave through an objective lens, the beam undergoes multiple light scattering as it propagates through the skull and this diffused light may activate optigenetic signals in all cells in an uncontrolled manner (left). By shaping the wavefront of the impinging beam using SLM, optical focus can be generated at the plane of the cells, thus activating optogenetic regulation at the level of individual cells (right). (b) Experimental setup, L1–3 lens: The blue dashed box indicates a wavefront shaping part, and the red dashed box indicates a fluorescence-imaging part. (c) Confocal image of second harmonic generation signals in the used mouse skull on the x-y plane (left) and in the axial direction (right). Scale bar 300 μm. (d) Intensity images of the transmitted beam through the skull without wavefront shaping (left), and with the wavefront shaping (right). (e) Measured intensity enhancements according to segment numbers and samples. (f) Measured intensity of optimized foci (n = 5) and intensity enhancements (n = 5) according to numbers of skull layers. The graph inset illustrates the optical transmittance according to numbers of skull layers. The circles represent experimental data, and a dashed line is a theoretically expected from Eq. (1). (g) Measured transport mean free path of the frontal (n = 4) and parietal bones (n = 4), respectively. Schematic of a mouse skull (top) and corresponding transport mean free paths of the mouse skull (bottom).

Mentions: In order to demonstrate that the wavefront shaping of excitation beams enables the generation of selective focus through a highly scattering skull layer and thereby the optogenetic regulation of signaling-pathways in individual cells, we used an in vitro model system in which a spatial light modulator (SLM) was employed for wavefront shaping (Fig. 2a). The experimental setup consists of two parts: the wavefront shaping part for activating light-sensitive proteins and the fluorescence imaging part for measuring cellular responses (Fig. 2b). To demonstrate the capability of optogenetic control through scattering tissues, a skull layer dissected from a mouse without a thinning procedure was prepared. The dissected skull layer was then attached to the bottom of a culture plate in which cells were dispersed in a monolayer. Thickness of the dissected skull was approximately 300 μm, measured from the second harmonic generation image (Fig. 2c). This was thick enough to scramble the incident laser beam into speckle patterns.


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)

Optical focusing through a skull layer using wavefront shaping technique.(a) Schematic of the experiment. Cells expressing both optoFGFR1 and R-GECO1 are placed above the skull layer. When illuminating with a plane wave through an objective lens, the beam undergoes multiple light scattering as it propagates through the skull and this diffused light may activate optigenetic signals in all cells in an uncontrolled manner (left). By shaping the wavefront of the impinging beam using SLM, optical focus can be generated at the plane of the cells, thus activating optogenetic regulation at the level of individual cells (right). (b) Experimental setup, L1–3 lens: The blue dashed box indicates a wavefront shaping part, and the red dashed box indicates a fluorescence-imaging part. (c) Confocal image of second harmonic generation signals in the used mouse skull on the x-y plane (left) and in the axial direction (right). Scale bar 300 μm. (d) Intensity images of the transmitted beam through the skull without wavefront shaping (left), and with the wavefront shaping (right). (e) Measured intensity enhancements according to segment numbers and samples. (f) Measured intensity of optimized foci (n = 5) and intensity enhancements (n = 5) according to numbers of skull layers. The graph inset illustrates the optical transmittance according to numbers of skull layers. The circles represent experimental data, and a dashed line is a theoretically expected from Eq. (1). (g) Measured transport mean free path of the frontal (n = 4) and parietal bones (n = 4), respectively. Schematic of a mouse skull (top) and corresponding transport mean free paths of the mouse skull (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4543936&req=5

f2: Optical focusing through a skull layer using wavefront shaping technique.(a) Schematic of the experiment. Cells expressing both optoFGFR1 and R-GECO1 are placed above the skull layer. When illuminating with a plane wave through an objective lens, the beam undergoes multiple light scattering as it propagates through the skull and this diffused light may activate optigenetic signals in all cells in an uncontrolled manner (left). By shaping the wavefront of the impinging beam using SLM, optical focus can be generated at the plane of the cells, thus activating optogenetic regulation at the level of individual cells (right). (b) Experimental setup, L1–3 lens: The blue dashed box indicates a wavefront shaping part, and the red dashed box indicates a fluorescence-imaging part. (c) Confocal image of second harmonic generation signals in the used mouse skull on the x-y plane (left) and in the axial direction (right). Scale bar 300 μm. (d) Intensity images of the transmitted beam through the skull without wavefront shaping (left), and with the wavefront shaping (right). (e) Measured intensity enhancements according to segment numbers and samples. (f) Measured intensity of optimized foci (n = 5) and intensity enhancements (n = 5) according to numbers of skull layers. The graph inset illustrates the optical transmittance according to numbers of skull layers. The circles represent experimental data, and a dashed line is a theoretically expected from Eq. (1). (g) Measured transport mean free path of the frontal (n = 4) and parietal bones (n = 4), respectively. Schematic of a mouse skull (top) and corresponding transport mean free paths of the mouse skull (bottom).
Mentions: In order to demonstrate that the wavefront shaping of excitation beams enables the generation of selective focus through a highly scattering skull layer and thereby the optogenetic regulation of signaling-pathways in individual cells, we used an in vitro model system in which a spatial light modulator (SLM) was employed for wavefront shaping (Fig. 2a). The experimental setup consists of two parts: the wavefront shaping part for activating light-sensitive proteins and the fluorescence imaging part for measuring cellular responses (Fig. 2b). To demonstrate the capability of optogenetic control through scattering tissues, a skull layer dissected from a mouse without a thinning procedure was prepared. The dissected skull layer was then attached to the bottom of a culture plate in which cells were dispersed in a monolayer. Thickness of the dissected skull was approximately 300 μm, measured from the second harmonic generation image (Fig. 2c). This was thick enough to scramble the incident laser beam into speckle patterns.

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.


Related in: MedlinePlus