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1.
Fig. 3.

Fig. 3. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Spatial resolution of TEFO activation of ChR2. (A) Stack image of a CA1PC loaded with Alexa594, showing location of laterally moved TEFO activation spots. (B) Lateral localization of response induced by 488-nm light (blue, n = 4) and TEFO (red, n = 3). (C) Axial localization of response induced by 488-nm light (blue) and TEFO with 50% (≈130 mW, red) and 100% (≈260 mW, black) power (dashed lines indicate Gaussian fit). (Inset) Individual responses at the focal plane of the dendrite (color-coded respectively). (D) Representative responses to 488-nm (blue) and TEFO-evoked (red) responses at different distances above and below the dendrite.

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.
2.
Fig. 5.

Fig. 5. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Fast and reliable AP responses by TEFO ChR2 excitation. (A) ChR2-mediated depolarization efficiently evoked APs in CA1 PV-INs (n = 3) and pyramidal cells (PC) in CA1 (n = 6) and subiculum (n = 4). ChR2-induced depolarization was measured by hyperpolarizing the soma as necessary to prevent AP firing. Graphs indicate the difference between resting membrane potential and AP threshold (green circles), amplitude of ChR2-induced suprathreshold depolarization (red squares), and the corresponding pulse duration (black squares) for each cell type. (B) Action potentials evoked in ChR2-expressing PV-IN by 1-ms-long pulses at 20–100 Hz. Insets: Subthreshold responses at hyperpolarized holding potential. Similar results were obtained in n = 3 cells. (C and D) Representative responses of a CA1PC (C, n = 7) and a subiculum PC (D, n = 6) to 20-, 50-, or 100-Hz stimulation with different pulse durations using single or multiple spots with different illumination time.

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.
3.
Fig. 6.

Fig. 6. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Large inhibitory synaptic terminals are activated by TEFO ChR2 excitation. (A) Representative traces of optically evoked hyperpolarizing postsynaptic potentials in a CA1PC innervated by ChR2-expressing PV-INs (red, TEFO excitation; blue, 488-nm excitation) by targeting the soma. SR-95531 (10 μM) blocked responses with both types of stimulations (black traces, representative for n = 3 cells). (B) Peak hyperpolarization evoked by TEFO (red) did not depend on pulse duration (n = 6). Excitation at 488 nm (blue) evoked larger responses than TEFO excitation (n = 5). Dots and error bars represent means ± SEM. (C) Stack image showing a recorded CA1PC and indicating locations of series of TEFO illumination spots at the soma (5 spots), stratum pyramidale (Pyr; 10 spots), stratum radiatum (Rad; 10 spots), and at the soma of a PV-IN (IN; 5 spots). (D) Representative traces showing responses to illumination at the locations indicated in C, using 100-ms stimulus intervals (green), 0.1-ms stimulus intervals (red), and 0.1-ms stimulus intervals in the presence of 0.5 μm TTX (black). (E) Overlaid traces evoked by stimulation at the soma, in stratum pyramidale (Pyr), and at the soma of the PV-IN (IN). Note the differences in response latency and the stepwise rise of the response evoked in stratum pyramidale.

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.
4.
Fig. 4.

Fig. 4. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Dependence of induced depolarization on tissue depth. (A) Dependence of induced depolarization at the somata of CA1PCs from Thy1-ChR2-YFP mice on tissue depth and spatiotemporal patterning of TEFO excitation for three different pulsed illumination durations and interpulse times. Red, single 1-ms pulses (n = 6, fitted linearly); open black dot and squares (total n = 3), single submillisecond pulses; filled circle and black dots (total n = 3), 5× 0.1-ms spatiotemporally patterned excitation. Note that repeating the 0.1-ms pulses five times on the same cells (filled black dot and squares, total excitation time ≤1 ms) yields a higher total depolarization compared with the single-spot 1-ms excitation. This can be used to induce bigger depolarizations at larger tissue depths. (B) Ten pulses (0.1 ms long) repeated three times with 0.1-ms intervals at the indicated locations in the apical tuft (distance from soma ≈400 μm, depth in slice ≈150 μm) evoked sufficient depolarization to evoke somatic AP (upper trace), whereas responses to single activations were almost undetectable (bottom traces).

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.
5.
Fig. 2.

Fig. 2. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Temporal resolution of TEFO activation of ChR2 in hippocampal neurons of acute brain slices. (A) Representative traces and summary graph (B) (n = 5) of depolarization evoked by 1- to 100-ms-long TEFO pulses at the somata of hippocampal neurons. (A, Inset) Representative response to 488-nm light. (C) Single focal section of a ChR2-expressing CA1PC loaded with Alexa594 in hippocampal slice from Thy1-ChR2-YFP mouse, with numbered TEFO illumination spots indicated by red dots. Bottom traces show membrane potential responses to 2-ms-long stimulations at the spots indicated in C. Note that somatic stimulation fires the cell reliably, whereas dendritic stimulation results in smaller depolarization, and stimulation at a spot outside of the cell area has no effect. (D) CA1PC soma with five two-photon temporal focusing spots indicated by dots. (Lower) Response to 1-ms-long TEFO excitation at a single spot (black) as well as to 0.1-ms-long two-photon temporal focusing illuminations at three to five spots with 0.1-ms intervals. Individual excitations are also shown (Lower, interval = 300 ms). Note that scale bar for the uppermost trace differs from that related to the four lower traces. Depolarization was more effective with multiple short illuminations than that obtained with a single 1-ms pulse, despite the shorter total excitation time.

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.
6.
Fig. 1.

Fig. 1. From: Two-photon single-cell optogenetic control of neuronal activity by sculpted light.

Characterization of the biophysical properties of TEFO activation of ChR2 in HEK293 cells. (A) Superimposed fluorescent and differential interference contrast (DIC) image of a HEK293 cell during recording. (B) Representative traces of induced inward current by one-photon excitation of ChR2 (blue), TEFO excitation of ChR2-expressing (red), and of control uninfected cell (black), using 100-ms-long pulses. (C) Representative traces of TEFO-activated ChR2-mediated inward current with different pulse durations. (D and E) Peak current amplitude (D) and rise time (E) at different wavelengths measured as a function of average power at the sample for a ≈3.8-μm spot and 100-ms duration (n = 22 cells). Inset in D shows the nonlinear dependence of peak inward current on the power at lower power levels, which is the result of two-photon excitation of ChR2. (F) Wavelength-dependency of TEFO excitation of ChR2 (n = 16 cells). Inset in F emphasizes the differences in activation efficiency of 800-nm and 880-nm light. (G and H) Peak current (G) and rise time (H) as a function of spot size at 880-nm wavelength and 100-ms duration (n = 13 cells). Inset in G shows the induced peak current with the TEFO for a constant photon number (constant power at 200 mW) for different spot sizes. Maximum amplitude and fastest rise time could be achieved with a spot size of ≈5–6 μm (I). Dots and error bars represent means ± SEM.

Bertalan K. Andrasfalvy, et al. Proc Natl Acad Sci U S A. 2010 June 29;107(26):11981-11986.

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