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, 95 (8), 3916-26

In-depth Activation of Channelrhodopsin 2-sensitized Excitable Cells With High Spatial Resolution Using Two-Photon Excitation With a Near-Infrared Laser Microbeam

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In-depth Activation of Channelrhodopsin 2-sensitized Excitable Cells With High Spatial Resolution Using Two-Photon Excitation With a Near-Infrared Laser Microbeam

Samarendra K Mohanty et al. Biophys J.

Abstract

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Figures

FIGURE 1
FIGURE 1
Confocal laser scanning imaging, activation, and detection of ChR2-expressing HEK cells. (a) ChR2 expression identified by YFP imaging (using a 20× objective) of cells not incubated with ATR. (b) Basal level of Ca orange fluorescence of cells without ATR. (c) Ca orange fluorescence subsequent to irradiation with the scanning IR two-photon laser microbeam (954 nm at power density of ∼1 × 106 mW/mm2). The encircled cell shows high YFP fluorescence but no significant base level of Ca dye fluorescence (excited by 543 nm) or enhancement with the IR two-photon laser microbeam (954 nm). (d) YFP imaging of ChR2-expressing cells incubated with ATR. (e) Increase in Ca orange fluorescence subsequent to selected activation with the scanning IR two-photon laser microbeam (954 nm) in a rectangular band. (f) Whole-field activation with the single-photon laser microbeam (477 nm, 100 mW/mm2). (g) Repeated irradiation with the NIR two-photon laser microbeam (954 nm at a power density of ∼1 × 107 mW/mm2) led to damage of the cell membrane (in the rectangular region) as indicated by a decrease in YFP fluorescence as well as (h) basal Ca orange fluorescence. (i) Irradiation with the NIR two-photon laser microbeam (954 nm at a power density of ∼1 × 106 mW/mm2) led to activation (indicated by increase in Ca orange fluorescence) in the intact cells.
FIGURE 2
FIGURE 2
Single-photon activation spectrum determined by mapping increase in Ca orange fluorescence (excited with a 543 nm laser beam at the same point) subsequent to irradiation with a visible cw laser activating the microbeam (focused to a radius of ∼0.55 μm using a 20× objective). (a) Decay of Ca fluorescence by bleaching at a single excitation point. (b) Change in Ca orange fluorescence from a point in the plasma membrane on exposure to 458 nm, (c) 477 nm, and (d) 488 nm. (e) No detectable Ca fluorescence was observed on activation with 514 nm. In a, the red graph represents the fluorescence excitation beam (543 nm), and in be it represents scattered activation laser beams detected through a separate channel. (f) Single-photon activation spectrum obtained from measurements over 25 points on five different cells. The power density of the activating laser beam was kept at ∼100 mW/mm2.
FIGURE 3
FIGURE 3
Mapping increase in Ca orange fluorescence subsequent to NIR fs laser microbeam irradiation (focused to a radius of ∼1.1 μm using a 20× objective). (a) Very small change in Ca orange fluorescence (excited with 543 nm laser beam at the same point) from a point in the plasma membrane, on exposure to the 860 nm laser microbeam. Increasing change in Ca orange fluorescence observed on exposure to (b) 880 nm and (c) 916 nm. Ca orange fluorescence was found to decrease on exposure to (d) 954 nm and (e) 976 nm, and is almost undetectable at 1028 nm (f). The average power density of the activating laser beam was kept at ∼1.0 × 106 mW/ mm2. The red graphs in a and b represent scattered activation laser beams detected through a separate channel. In c–f, the pulses of scattered activation at longer wavelengths could not be detected due to technical limitations. (g) Two-photon activation spectrum determined from measurements over 30 points on six different cells.
FIGURE 4
FIGURE 4
Dependence of Ca orange fluorescence (excited with a 543 nm laser beam at the same point on the plasma membrane) on the power density of the two-photon laser microbeam (916 nm, focused to a radius of ∼1.1 μm using a 20× objective): (a) 3.9 × 105, (b) 7.8 × 105, (c) 11.7 × 105, and (d) 15.6 × 105 mW/mm2. (e) Fluorescence intensity (as a measure of activation) as a function of power density of the two-photon laser microbeam, determined from measurements over 20 points on four different cells. The pulses of scattered activation at longer wavelengths could not be detected due to technical limitations.
FIGURE 5
FIGURE 5
Comparison of in-depth activation efficacy (measured by the maximum depth from which Ca orange fluorescence is detected) of ChR2-expressing neuronal cells in mouse hippocampus. (a) ChR2-expression observed by YFP imaging. (b–e) Basal level of Ca orange fluorescence at depths of 180, 200, 220, and 240 μm, respectively. (f–j) Single-photon activation with a laser microbeam (458 nm, 1.0 × 103 mW/mm2). (k–o) Activation with 477 nm. (p–t) Two-photon irradiation with the NIR laser microbeam (916 nm) leading to an increase in Ca orange fluorescence at all depths. Histogram of fluorescence intensity (as a measure of activation efficiency) versus wavelength of activation at depths of 160 μm (u), 180 μm (v), 200 μm (w), 220 μm (x), and 240 μm (y). Scale bar in a represents 200 μm.

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