Supplementary moviecaptions and figures:
Suppl. movie 1: Wide-area activation of ChR2 expressing HEK cells with scanning laser microbeam as noticed by increase in calcium orange fluorescence.
Suppl. movie2:Non-homogenous expression ofChR2 visualized by confocal imaging of marker YFP fluorescence (green) from different regions of the HEK cells. Red fluorescence is from calcium orange staining.
Suppl. Fig.1:Patch clamp recording from ChR2-expressing cells (identified by fluorescence microscopy) subjected to light assisted activation.
Suppl. Fig.2:Kinetics of calcium orange fluorescence from the focused region of the ChR2-expressing cell membrane irradiated with the near infrared fs pulse train (100 ms, 916 nm, 97.64 MHz).
Suppl. Fig.3: Non-overlapping excitation spectrum of ChR2 (orange dashed line), YFP (blue dashed line) and Calcium orange (green dashed line). Selected detection bands (shown by solid regions) from emission spectrum of the YFP (blue solid line) and calcium indicator dye (green solid line).
Suppl. Fig.4: Instantaneous rise in calcium influx (red arrow) persisting for quite long period indicating optoporation of calcium indicator dye into HEK cells at laser microbeam threshold power density of ~0.5 x 107 mW/mm2 (100 ms exposure).
Suppl. Fig.5: Comparison of in-depth activation efficacy (measured by maximum depth from which calcium orange fluorescence is detected) of ChR2-expressing HEK cells. (a) Three dimensionally reconstructed image of ChR2-expressing (identified by YFP imaging) HEK cells growing as a spheroid cluster. (b) XZ cross-section of the fractional-spheroid of ChR2-expressing HEK cells. Single photon activation with laser microbeam (1.0 x 103 mW/mm2) leading to an increase in calcium orange fluorescence over a part of the fractional-spheroid at (c) 458nm, (d) 477nm and (e) 488 nm. (f) Two-photon irradiation with near IR laser microbeam (954 nm, 1.0 x 106 mW/mm2) leading to activation over the whole three-dimensional structure. Scale bar in a represents 300m.
Suppl. Fig.1:Patch clamp recording from ChR2-expressing cells (identified by fluorescence microscopy) subjected to light assisted activation.
Suppl. Fig.2:Kinetics of calcium orange fluorescence from the focused region of the ChR2-expressing cell membrane irradiated with the near infrared fs pulse train (100 ms, 916 nm, 97.64 MHz).
Suppl. Fig.3: Non-overlapping excitation spectrum of ChR2 (orange dashed line), YFP (blue dashed line) and Calcium orange (green dashed line). Selected detection bands (shown by solid regions) from emission spectrum of the YFP (blue solid line) and calcium indicator dye (green solid line).
Suppl. Fig.4: Instantaneous rise in calcium influx (red arrow) persisting for quite long period indicating optoporation of calcium indicator dye into HEK cells at laser microbeam threshold power density of ~0.5 x 107 mW/mm2 (100 ms exposure).
Suppl. Fig.5: Comparison of in-depth activation efficacy (measured by maximum depth from which calcium orange fluorescence is detected) of ChR2-expressing HEK cells. (a) Three dimensionally reconstructed image of ChR2-expressing (identified by YFP imaging) HEK cells growing as a spheroid cluster. (b) XZ cross-section of the fractional-spheroid of ChR2-expressing HEK cells. Single photon activation with laser microbeam (1.0 x 103 mW/mm2) leading to an increase in calcium orange fluorescence over a part of the fractional-spheroid at (c) 458nm, (d) 477nm and (e) 488 nm. (f) Two-photon irradiation with near IR laser microbeam (954 nm, 1.0 x 106 mW/mm2) leading to activation over the whole three-dimensional structure. Scale bar in a represents 300m.