Holographic photolysis for multiple cell stimulation in mouse hippocampal slices

Abstract

Background: Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.

Methods/principal findings: The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca(2+) imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.

Conclusions/significance: We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.

Figure 1. Optical set-up for holographic illumination.

Layout of the optical set-up. The focal length of the lenses are f₁ = 750 mm, f₂ = 500 mm, f₃ = 200 mm, f₄ = 75 mm. See methods for details.

Figure 2. Optical sectioning with HiLo microscopy.

A–C. Speckle (A) and uniform (B) illumination images used in the calculation of the quasi-confocal HiLo image (C). D. z-projection of 12 HiLo sections (including C) separated by Δz = 2 µm. E. Measurement of the HiLo microscope axial resolution: integrated signal from a thin fluorescent layer (≈0.3 µm) as a function of defocus z. The measured axial resolution is 4 µm FWHM.

Figure 3. Holographic multiple cell photostimulation.

A. Overlay of a fluorescence image recorded with HiLo microscopy showing OGB-loaded CA1 neurons of a hippocampal slice with three different excitation pattern configurations for uncaging: elliptic, shaped (cell somata) and anti-shaped (extracellular space) patterns. The superimposed red images of the excitation spots were obtained by exciting a thin layer of fluorescein. B. Measured y-z intensity cross-sections of the excitation beam along the yellow lines shown in A. The contour lines of OGB-loaded cells imaged with HiLo microscopy are superimposed (white lines). C. Distributions of calculated (red) and measured (black) axial intensities for the spots in A. The calculation was based on Angular Spectrum of Planar Wave Approximation algorithm .

Figure 4. Electrophysiological recordings upon holographic photostimulation of multiple neurons.

A. Images of three pattern configurations obtained by exciting a thin layer of fluorescein: ellipse (a), shaped (b) and anti-shaped (c) patterns. Scale bar: 20 µm. B. Photolysis-evoked currents elicited in a recorded CA1 neuron held at −60 mV by uncaging MNI-glutamate in a group of target cells with the three patterns in (A), using an energy of 3.5 µJ at the sample plane. Individual sweeps (gray) and averaged currents (black) are shown (excitation densities: 0.9 nJ/µm², 3.6 nJ/µm² and 10 nJ/µm² for the elliptic, shaped and anti-shaped patterns, respectively). C. Histogram of current amplitude increases obtained with shaped and anti-shaped patterns normalized by the currents obtained with an ellipse. D. Mean normalized amplitudes of photolysis-evoked currents obtained in five recorded neurons held at −60 mV and excited with a shaped pattern, while changing the focal plane of the objective with steps of 3 µm. The top of the slice is indicated by the black dashed line. The theoretical axial distribution of light (I_A) for a shaped pattern is superimposed. Scale bar for the inset: 10 µm. E. Photolysis-evoked responses recorded in the same cell as in (B) in current-clamp mode. F. Comparison of spiking rates obtained with the three pattern configurations in four distinct cells.

Figure 5. Ca²⁺ imaging upon holographic photostimulation of multiple neurons.

A. (left) Fluorescence images obtained with HiLo microscopy showing CA1 neurons of a hippocampal slice. (right) Scheme of responding (red) and non-responding (black) cells to photostimulation with a shaped pattern (blue). B. Images of the three patterns used to photostimulate target neurons in CA1 with MNI-glutamate using the same energy of 3.6 µJ, corresponding to 1.7 nJ/µm², 6.2 nJ/µm² and 18.1 nJ/µm² for the elliptic, shaped and anti-shaped patterns, respectively. C. Variation of intracellular Ca²⁺ concentration of two different target neurons during photostimulation with the three different pattern configurations.

Figure 6. Holographic photostimulation of NG2 cells.

A. Red fluorescence image of a target DsRed⁺ NG2 cell (arrowhead) in a hippocampal slice of NG2-DsRed transgenic mice. B. Photolysis-evoked currents elicited in the same cell held at −70 mV upon illumination with a 5 µm spot (a) and shaped pattern (b), using the same power density (120 nJ/µm²). Individual sweeps (gray) and averaged currents (red and black) are shown. C. Normalized photolysis-evoked currents shown in (B). Note the similarity of the kinetics. D. Fluorescence images of a target DsRed⁺ NG2 cell in a OGB-loaded hippocampal slice of NG2-DsRed transgenic mice (arrowheads). E. Variation of intracellular Ca²⁺ concentration during photostimulation with a 5 µm spot (a) and shaped pattern (b), using the same power density (100 nJ/µm²). Scale bars for insets: 10 µm.