Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning

Abstract

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.

Fig. 1.

Combined remote-focusing and digital holography setup. Purple path, digital holography. Blue path, epifluorescence. Green path, remote-focusing system generating a remote image of the sample (solid line), by imaging the pupil plane of O1 into that of O2 (dashed line). S, sample; O1, objective Olympus, water immersion, 40×, 0.8 N.A.; O2, objective Olympus, dry, 40×, 0.75 N.A.; L1-L5, achromatic lenses [focal lengths (millimeter), f_L1 = 200; f_L2 = 150; f_L3 = 150; f_L4 = 300; f_L5 = 500]; P, polarizing beam combiner; λ/4, quarter wavelength plate; M_R, remote mirror; SLM, spatial light modulator. (Insets) Formation of the remote image (Img) and of the mirror image (Img’, red), following axial translation (Top) or tilting (Bottom) of M_R.

Fig. 2.

Characterization of defocus and spherical aberrations in the remote-focusing setup. (A) Optical setup used for characterization of z-dependent aberrations introduced by a single objective (O1 or O2). M_O, mirror in object space; L1, L2 achromatic lenses [focal lengths (millimeter), f_L1 = 200; f_L2 = 100]; C, 50/50 beam splitter cube. (B) Zernike coefficients for (Top to Bottom) defocus (), first-order (), and second-order () spherical aberrations, measured with the setup of A, experimental data (blue triangles and black squares, for O1 and O2, respectively) were in agreement with theoretical predictions (blue and black lines, for O1 and O2, respectively) obtained in the linear approximation for N.A._O1 = 0.76 and N.A._O2 = 0.72. (C) Remote-focusing setup modified for aberration measurements. L1-L4, achromatic lenses [focal lengths (millimeter), f_L1 = 200; f_L2–L3 = 150; f_L4 = 100]. (D, Top and Middle). Zernike coefficients for defocus () and first-order spherical aberration () introduced by O1 (blue triangles), O2 (black squares) were measured in the setup of C by moving M_O or M_R, respectively. Then, defocus was canceled (Top, red circles) by moving M_O and M_R simultaneously. First-order spherical aberration was almost perfectly compensated (Middle, red circles) and could be well approximated by summing (red line) the linear fits of first-order spherical aberrations introduced by O1 and O2 (blue and black lines, respectively), in agreement with theory. (Bottom) Measured Strehl ratio for uncompensated (blue triangles) and compensated (red circles) configurations. The green star corresponds to the Strehl ratio measured in setup A with objective O1 (without the remote-focusing unit).

Fig. 3.

Imaging along a titled plane. (A) Schematic of the incoming (solid blue line) and reflected (dotted blue line) beam at O2, with tilted M_R (Bottom, side view; Top, pupil plane): α₂, semiaperture angle of the incoming beam; β, semiaperture angle of O2; γ, M_R tilt angle with respect to the focal plane of O2 (corresponding to a tilt of the remote image twice larger). If γ exceeds the value of (β-α₂)/2, part of the reflected beam is clipped by the pupil of O2, leading to resolution loss. (B) Strehl ratio measured for different values of 2γ (tilt of the remote image) with respect to the horizontal-axis (x₂). The setup was arranged as in Fig. 2C, but with f_L4 = 50 mm. (C) CA1 pyramidal neuron filled with OGB-1, imaged before (Top) and after (Bottom) tilting M_R to compensate for the inclination of the apical dendrite to the surface of the slice (10°). Before tilting the mirror, only a small portion of the apical dendrite was in-focus (dashed red line), whereas the whole extent of the dendrite in the field-of-view (∼200 μm) was in-focus after tilting. Scale bar, 10 μm.

Fig. 4.

Independent 3D photostimulation and Ca²⁺ imaging along a tilted plane in CA1 pyramidal neurons filled with OGB-1. (A) Schematic of the uncaging configuration corresponding to B–E: MNI-glutamate photolysis was performed at three locations along the tilted apical dendrite (red arrows, z = -10.5; 0, 7.5 μm for the proximal, medial and distal uncaging locations, respectively). (B and C) Images of the neuron before and after tilting the remote mirror to follow the direction of the principal dendrite in the brain slice (12°). (D) Ca²⁺ response (ΔF) measured 100 ms after photoactivation. The full time course is available as Movie S1. (E) Ca²⁺ responses (ΔF/F) measured for different ROIs along the dendrite (1–8, cyan lines) at 10 Hz (Left) and 60 Hz (Right) acquisition rate. The ΔF movie at 60 Hz is available as Movie S2. (F) Schematic of the uncaging configuration corresponding to panels G–K: MNI-glutamate photolysis is performed at three locations on a thin branch off the apical dendrite (red arrows; z = 18, 22.5, 34.5 μm). (G) Fluorescence image of the neuron schematized in F, at the focal plane (z = 0). (H) Maximum intensity projection obtained from a z-stack of the neuron (40 frames; Δz = 1.5 μm), indicating the position of the uncaging spots, which are axially displaced with respect to the tilted imaging plane (full stack available as Movie S4). (I) Image of the neuron after tilting the imaging plane by 11° with the remote mirror. (J) Ca²⁺ response (ΔF) measured 100 ms after photolysis. The full time course movie is available as Movie S5. (K) Ca²⁺ responses (ΔF/F) measured from three ROIs along the apical dendrite (I, 1–3 cyan lines) at 10 Hz. Scale bars, 10 μm.

Fig. 5.

Reduction in the spatial spread of dendritic Ca²⁺ signals by block of voltage-dependent Na⁺ channels. (A) In the same CA1 pyramidal neuron, Ca²⁺ responses (ΔF) measured 200 ms after photolysis of MNI-glutamate at three locations (red arrows), in the absence (Left) or presence (Right) of 0.5 μM TTX. The cell was filled with OGB-1. (B) Pooled data representing Ca²⁺ signals (ΔF/F) averaged over different cells in the presence (red) or absence (black) of TTX. Thirty-five micrometers line profiles (Insets, black lines) were taken along the dendrite, centered on the medial (Top) and distal (Bottom) uncaging positions. Data are reported as mean ± SEM. Fluorescence signals (ΔF/F) were normalized to its maximum for each cell before averaging. The average power was 4.7 mW, both in control (4.7 ± 0.9 mW) and in TTX (4.7 mW for all cells). (C) Histogram reporting mean ± SEM values of normalized ΔF/F at ± 17 μm from the uncaging location. The level of Ca²⁺ signal was significantly smaller in the presence of TTX for the distal (D) and medial (M) uncaging locations, both in the forward (F) and in the backward (B) directions (DB, N_ctrl = 3, N_TTX = 3, p = 0.04; DF, N_ctrl = 3, N_TTX = 4, p = 0.006; MB, N_ctrl = 4, N_TTX = 4, p = 0.003; MF, N_ctrl = 4, N_TTX = 4, p = 0.0003). N values are identical for B and C. Laser pulse duration: 1 ms. Scale bar, 10 μm.