2.1.

Slice Preparation, Cell Loading, and Recording

We prepared slices of somatosensory cortex from wild-type mice (C57BL/6J) in accordance with the guidelines of European Union and institutional guidelines of the care and use of laboratory animals (Council directive 86/609 European Economic Community). Male or female mice (3 to 4 weeks of age) were deeply anesthetized with isoflurane (5%) and killed through decapitation. The brain was rapidly removed and placed in ice-cold ($<5°\mathrm{C}$) or room temperature cutting solution containing the following (in mM): 110 choline chloride, 2.5 KCl, 7.0 ${\mathrm{MgCl}}_2$, 0.5 ${\mathrm{CaCl}}_2$, 25 ${\mathrm{NaHCO}}_3$, 1.25 ${\mathrm{NaH}}_2{\mathrm{PO}}_4$, 20 glucose, and aerated with 95% ${\text{O}}_2$, 5% ${\mathrm{CO}}_2$ to a final pH of 7.4. We dissected the cerebral cortex, blocking it to take coronal slices of somatosensory cortex. The cortical block was glued to an ice-cold stage on a Leica microslicer, and $300\mu \mathrm{m}$ slices were cut in ice-cold cutting solution. Cut sections were placed in an incubator at 35°C for 0.5 h in the cutting solution in a chamber containing ACSF (in mM): 125 NaCl, 2.5 KCl, 1.0 ${\mathrm{MgCl}}_2$, 2.0 ${\mathrm{CaCl}}_2$, 25 ${\mathrm{NaHCO}}_3$, 1.25 ${\mathrm{NaH}}_2{\mathrm{PO}}_4$, 20 glucose, and 0.4 L-ascorbic acid. Slices were kept at room temperature for at least 0.5 h before dye loading and recording in a submersion chamber.

2.2.

Dye Loading

We visualized recovered cortical slices with Olympus BX51WI’s native infrared differential-interference contrast (IR-DIC) path, further magnifying the image onto a scientific complementary metal oxide semiconductor (sCMOS, ORCA Flash 4, Hamamatsu) camera with a $2\times /4\times$ magnification changer (Fig. 1 “MC”; Luigs and Neumann, Ratingen, Germany) mounted above the tube lens. We loaded neurons with VSD with a glass patch electrode by filling the tip with dye-free internal solution (in mM: 130 K-gluconate, 7 KCl, 4 ATP-Mg, 0.3 GTP-Na, 10 phosphocreatine-Na, 10 HEPES; adjusted to pH 7.4 and $284\mathrm{mol}/\mathrm{kg}$), then back-filling with the dye-containing internal solution (JPW3028, 400 to $800\mu \mathrm{M}$), and performed whole-cell somatic recordings from the selected neuron under DIC optics. The dye JPW3028 is a doubly positively charged analog of the aminostyryl-pyridinium series of lipophilic VSDs available from Invitrogen as D-6923. This electrochromic dye does not significantly increase the membrane capacitance of the labeled neuron, as evident from several control measurements showing that the waveform of the electrically recorded action potentials remains unaltered after intracellular dye loading.¹⁶ Dye-free solution in the tip is necessary because this highly lipophilic molecule would otherwise spill onto the slice before sealing and patching the cell of interest, as any dye bound to membrane outside the cell of interest increases the background fluorescence and thus degrades recording sensitivity. The cell was patched and stained at room temperature for 15 to 30 min by allowing passive diffusion of the dye into the cell. The dye diffuses slowly compared with other dyes of the same size and weight due to its high affinity for lipid membranes. After staining, we carefully detached the patch electrode from the neuron, forming an outside-out patch, after which the slice was incubated at room temperature for 1 to 2 h, allowing the dye to diffuse from the soma into the axon and neighboring basal dendrites.

Fig. 1

Computer-generated holography (CGH) schematic and axial propagation. (a) The beam emitting from the solid-state 532 nm, 450 mW laser (LS) is attenuated with a neutral density filter (ND), a half-wave plate ($\lambda /2$) and polarizing beam splitter (BS), reading and adjusting the power with removable mirror (M2), and a power meter (PM). Lenses ${\mathrm{L}}_{\mathrm{BE}1}$ ($f=19\mathrm{mm}$) and ${\mathrm{L}}_{\mathrm{BE}2}$ ($f=150\mathrm{mm}$), and pinhole (PH, $d=15\mu \mathrm{m}$) clean and expand the beam to fill the active area of the spatial light modulator. Lens ${\mathrm{L}}_1$ ($f=400\mathrm{mm}$) converts the modulated wavefront into a spatial light pattern, which is demagnified into the sample plane by a telescope formed by ${\mathrm{L}}_2$ ($f=200\mathrm{mm}$) and the objective ($\mathrm{NA}=1.1$, $M=60\times$, $f=5\mathrm{mm}$). The zero-order component is blocked by ${\mathrm{Bl}}_0$ before the beam enters the Olympus BX51WI microscope. The 532-nm fluorescence excitation light is reflected through the objective by a 560LP dichroic. Fluorescence transmitted by the dichroic is long-pass filtered (${\mathrm{F}}_{\mathrm{EM}}$, 593LP) and imaged onto a high-speed scientific-CMOS camera (sCMOS) by the native Olympus tube lens (${\mathrm{L}}_{\mathrm{TB}}$) and magnification changer (MC). (b) Scheme of large shaped “square with slot” illumination used for imaging dendrites and axons before voltage imaging measurement. The squared spot (100 μm) has a light-free region in the center (of around $30\mu \mathrm{m}$ width) to spare the dye filled soma from unnecessary illumination and photodamage. The lateral shape masks (green) are specified based on widefield epifluorescence images of voltage-sensitive dye (VSD) filled structures. (c) Simulated axial propagation profile of light shaped to the contours of the axons or dendrites of interest. Full width at half maximum (FWHM) $z$-confinement is $7.1\mu \mathrm{m}$. (d) Simulated axial propagation profile of large-spot “pseudowidefield” (pWF) illumination. FWHM $z$-confinement is $50.1\mu \mathrm{m}$.

2.3.

Computer-Generated Holography

Dye-loaded cells were illuminated with a 450 mW frequency-doubled diode-pumped $\mathrm{Nd}:{\mathrm{YVO}}_4$ low-noise laser emitting at 532 nm (Fig. 1 “LS”; MLL-FN-532-450-5-LAB-TTL, Changchun New Industries Optoelectronics Tech. Co. Ltd., Changchun, China) with CGH to achieve desired spatial patterns of light at the objective focal plane. Specifically, the laser beam (Fig. 1, LS) is attenuated with a neutral density (ND) filter, a half-wave plate ($\lambda /2$) and polarizing beam splitter, reading and adjusting the power with a removable mirror (M2), and a power meter (PM, Newport 818-ST2). Lenses ${\mathrm{L}}_{\mathrm{BE}1}$ ($f=19\mathrm{mm}$) and ${\mathrm{L}}_{\mathrm{BE}2}$ ($f=150\mathrm{mm}$), and pinhole (PH, $d=15\mu \mathrm{m}$) clean and expand the beam to fill the active area of the liquid crystal on silicon spatial light modulator (LCOS-SLM, Hamamatsu X10468-01). Lens ${\mathrm{L}}_1$ ($f=400\mathrm{mm}$) converts the modulated wavefront into a spatial light pattern, which is demagnified into the sample plane by a telescope formed by ${\mathrm{L}}_1$, ${\mathrm{L}}_2$ ($f=200\mathrm{mm}$) and the microscope objective (Fig. 1 “OBJ,” Olympus LUMFLN 60XW, $\mathrm{NA}=1.1$, $f=5\mathrm{mm}$). The 532-nm fluorescence excitation light is reflected through the objective by a 560-nm long-pass dichroic (FF560-FDi01-25x36, Semrock, Rochester, New York). Fluorescence transmitted by the dichroic is long-pass filtered (${\mathrm{F}}_{\mathrm{EM}}$, 593LP, FF01-593/LP-25, Semrock) and imaged onto a high speed scientific-CMOS camera (sCMOS, Hamamatsu ORCA Flash 4.0) or electron multiplying charge-coupled device (EMCCD, Andor 860 iXon3) by the native Olympus tube lens (${\mathrm{L}}_{\mathrm{TB}}$) and MC used to target patch clamp electrodes. In order to block the zero-order non phase-modulated component reflected from the SLM, we introduced a defocus in the beam by adjusting the distance between ${\mathrm{L}}_{\mathrm{BE}1}$ and ${\mathrm{L}}_{\mathrm{BE}2}$ in order to displace the zero-order focus by 30 to 40 mm after the Fourier plane of the first lens.^17,18 For the diffracted first order, the defocus was compensated with a spherical Fresnel lens at the SLM. Thus, with the zero-order displaced 30 to 40 mm from the effective Fourier plane of ${\mathrm{L}}_1$, we could block the unwanted zero-order component with a point block [Fig. 1(a), “${\mathrm{Bl}}_0$” tape on cover slip] without perturbing the propagation of the hologram (first-order beam). Phase holograms were calculated with an iterative Fourier transform algorithm (IFTA).^19–22 The IFTA-generated phase profiles were computed and addressed to the SLM using “Wavefront Designer IV,” in house software written in C++ with Qt 4.4.0 and fftw 3.1.2.²³

After the dye diffusion period, we imaged the neuron’s dendrites and axon at depths of 10 to $50\mu \mathrm{m}$, weakly illuminating (excitation density $\sim 1\mathrm{nW}/\mu {\mathrm{m}}^2$) with a CGH-generated square shape ($100\mu \mathrm{m}$) leaving a light-free slot in the center $\sim 30\mu \mathrm{m}$ wide [see Fig. 1(b)]. Positioning the soma in this light free slot enabled examination of axonal and dendritic structures, while sparing the dye-filled soma from unnecessary photodamage. Low-power density illumination was paired with long (500 ms) camera integration time and binning ($2\times 2$ or $4\times 4$) to maximize collection and minimize light exposure and photodamage to stained axons and dendrites during preimage acquisition. We then used fluorescence images of the axon and dendrites to define the spatial patterns of illumination for VSD signal acquisition trials, sculpted to the structure’s contours [Fig. 1(c)]. For VSD signal acquisition, we decreased the frame integration time to $156\mu \mathrm{s}$ ($\text{frame}\text{rate}=6.41\mathrm{kHz}$) and modulated the laser power to achieve an excitation density in the range of 0.6 to $10\mu \mathrm{W}/\mu {\mathrm{m}}^2$ across the axon- or dendrite-shaped region. Laser light was gated onto the cell with a high-speed shutter [Fig. 1(a)] “Sh”; Uniblitz LS6, driver D880C; Rochester, New York] for 10 to 30 ms trials, with 1 to 2 min intertrial intervals. During each trial, we stimulated action potentials with brief current pulses (5 to 10 ms, 400 to 800 pA) applied through a dye-free pipette patched in whole-cell configuration. Electrical and optical waveforms were monitored for significant changes in width and fall-time indicative of photodamage, at which point the experiment was discontinued. CGH-sculpted light illumination trials were interleaved with “pseudowidefield” (pWF) trials illuminated with a large diameter (25 to $40\mu \mathrm{m}$) spot with poor axial confinement [Fig. 1(d)], increasing laser power to maintain an excitation density equal to that of the sculpted light trials.

Current clamp signals were recorded at 20kHz with a MultiClamp 700B amplifier and 1440A digitizer (Molecular Devices, Sunnyvale, California), which also triggered camera acquisition and shutter opening. Micromanager²⁴ piloted the sCMOS and Andor Solis drove the EMCCD. We analyzed fluorescence signals during experiments with the ImageJ²⁵ time series plug-in and custom MATLAB scripts. Posthoc image visualization, processing, analysis, and statistics were performed in ImageJ and “VKAT: Voltage-imaging Kinetics Analysis Tool,” a wxpython-based GUI leveraging modules Numpy and Matplotlib.

2.4.

Axial Propagation Simulations

In order to estimate and compare the $z$-confinement of CGH shaped and “pWF” configurations, we simulated the distribution of the holographic beam propagating along the optical axis around the objective focal plane as described in Ref. 26. Briefly, we calculated the beam irradiance around the objective focal plane after the input phase hologram propagates through the telescope formed by ${\mathrm{L}}_1$, ${\mathrm{L}}_2$, and the objective using the angular spectrum approach of plane waves with a thin element approximation. Lutz et al.²⁶ demonstrated that the simulations faithfully predict the experimentally measured propagation of shaped, CGH-generated spots. Although these simulations do not factor in depth-dependent brain tissue scattering, Zahid et al.¹⁸ have shown that holographic spots maintain axial confinement at depths of $30\mu \mathrm{m}$ in hippocampal slices. The axial confinement of the simulated beam propagation was quantified as full width at half maximum (FWHM) of intensity averaged over the “dendrite shaped” region of interest (ROI) in each axial plane [Fig. 1(c)].

2.5.

Analysis

Axons and dendrites were discriminated visually, axons being thin and straight with collaterals issuing at obtuse angles, and dendrites thicker, spiny, and branching off in acute angle “Y”s. We spatially averaged fluorescence signals from each imaged axon or dendrite over groups of 12 to 18 pixels. Acquiring frames at 6410 Hz, our sCMOS camera operated in “rolling shutter” mode, resulting in a systematic exposure delay of each horizontal line on the chip. Any temporal distortion potentially induced by the rolling shutter was canceled by spatially averaging axon and dendrite ROIs over pixels located across similar numbers of horizontal lines. All data shown and reported come from single-trial measurements. We applied a low-pass binomial filter (1 to 2 passes) to reduce high-frequency noise. In order to improve the temporal precision of spike kinetic measurements, the data collected at $6410\text{frames}/\mathrm{s}$ were reconstructed using cubic spline interpolation, and then resampled to 100 kHz before measuring spike width and rise time. Both for shaped and pWF trials, spike kinetic measurements were taken from ROIs in which both CGH excitation and widefield imaging were parfocal with the structure of interest. Axons and dendrites occupied different axial planes preventing simultaneous widefield epifluorescence imaging of both structures. Axonal and dendritic data were thus collected in separate, alternate trials. Because the $\mathrm{d}F/F$ signal from different parts of the neuron varies owing to factors other than voltage (e.g., partitioning of dye in inner and outer membranes), changes in voltage indicated by changes in fluorescence could not be calibrated on an absolute scale. We therefore limited comparison of signals emanating from axonal and dendritic compartments to kinetic parameters that do not depend on precise, absolute voltage scale calibration.

Since variable nonspecific fluorescence precludes signal amplitude comparisons between different ROIs, we performed paired comparisons of baseline fluorescence, noise, fractional change, and S/N in compartments illuminated with shaped and large spot pWF in alternate trials. To quantify spike-evoked fluorescence amplitude, we calculated the fractional change in fluorescence $\mathrm{d}F$, with respect to the baseline fluorescence for each signal-bearing pixel. Specifically

We quantified differences between the kinetics (spike width, rise time) and S/N characteristics between axonal- and dendritic-generated waveforms, as well as CGH and “pWF” (large spot) configurations.

3.1.

Targeted Fluorophore Excitation Enables Signal Discrimination in Neighboring Structures

We found that CGH-shaped voltage dye fluorescence excitation in neighboring axons and dendrites enabled discrimination of differing action potential kinetics not possible with large-field illumination. Specifically, we compared 10% to 90% rise time and FWHM of action potential-evoked fluorescence transients recorded in axons and dendrites illuminated with CGH-sculpted shapes in alternate trials (Fig. 2). Corresponding to previously reported electrical^27,28 and optical measurements,^2,4,5 action potential rise time and FWHM were shorter [$p\text{-}\text{values}\le 0.05$ Student’s t-test; Figs. 2(b), 2(c), 2(e), and 2(g)] in axons (FWHM: $1.62\mathrm{ms}\pm 0.24$ standard error of the mean, S.E.M.; rise time: $522.1\mu \mathrm{s}\pm 95.6$ S.E.M., $n=3$ trials from two cells) than in dendrites (FWHM: $2.37\mathrm{ms}\pm 0.30$ S.E.M.; rise time: $1154.6\mu \mathrm{s}\pm 194.0$ S.E.M.; $n=4$ trials from two cells). Displayed action potential-evoked fluorescence transients emanating for the dendrite [Fig. 2(b)] and axon [Fig. 2(c)] were collected in separate trials since these structures occupied two different planes of focus. Figure 2(e) redisplays these two traces, amplitude normalized and peak aligned to show the difference in spike kinetics undetectable with pWF illumination [Fig. 2(a)]. Trials interleaved in which neighboring axons and dendrites were simultaneously illuminated with large spots did not show differences [Fig. 2(a), 2(f), and 2(g), $p\text{-}\text{values}>0.3$ Student’s t-test] in spike kinetics in the same axonal (FWHM: $1.87\mathrm{ms}\pm 0.09$ S.E.M.; rise time: $618.5\mu \mathrm{s}\pm 68.2$ S.E.M.; $n=4$ trials in two cells) and dendritic ROIs (FWHM: $1.80\mathrm{ms}\pm 0.15$ S.E.M.; rise time: $600.2\mu \mathrm{s}\pm 170.2$ S.E.M.; $n=3$ trials in two cells), even though these structures occupied different planes of focus.

3.2.

Targeted Excitation Increases Fractional Spike-Evoked Transient Amplitude but not Signal-To-Noise

Paired comparisons of CGH shaped ($n=4$) or “pWF” ($n=4$) trials revealed significantly decreased background fluorescence ($F_{\mathrm{Bkg}}=F_{\mathrm{BL}}-F_{\text{dark}}$, averaged over time postshutter opening and prespike), increased RMS noise and increased peak action potential evoked $\mathrm{d}F/F$ [$\mathrm{d}F/F_{\mathrm{MAX}}$ summarized by Fig. 3(table)]. Although inhomogeneous partitioning of the lipophyllic voltage dye precludes comparison of the signal amplitude between axonal and dendritic compartments, here we compare $\mathrm{d}F/F_{\mathrm{MAX}}$ emanating from the same ROIs under sculpted and pWF illumination conditions. Since both the signal ($\mu =\mathrm{d}F/F_{\mathrm{MAX}}$) and RMS noise increased with CGH-shaped excitation, we observed no change in S/N.

Fig. 3

Comparison of baseline fluorescence, signal noise, spike-waveform amplitude, and signal-to-noise ratio (S/N). The table shows mean and standard error of the mean for trials taken with CGH shaped VSD excitation and large-spot “pWF” excitation for: (1) background fluorescence ($F_{\mathrm{Bkg}}=F_{\mathrm{BL}}-F_{\text{dark}}$, postshutter opening and prespike) fluorescence in arbitrary units (A.U.) after dark (preshutter opening) value subtraction; (2) root-mean-square (RMS) noise of the prespike signal ($\%\mathrm{d}F/F$); (3) maximum $\mathrm{d}F/F$ (%) of the action potential-evoked fluorescence transient, and; (4) the S/N ratios. Paired Student’s t-tests indicate significant increases in RMS noise and peak $\mathrm{d}F/F$, and reduction baseline fluorescence between CGH shaped and pWF trials, illustrated as a percent change in the bar graph (lower). There was no significant difference in S/N between CGH shaped and pWF trials.