Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

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Abstract

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5 mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

Introduction

There has been a recent trend toward applying two-photon microscopy to image the activity of neuronal populations rather than individual neurons. However, because two-photon microscopy requires laser scanning, such imaging invariably involves a tradeoff between temporal resolution and field of view, or FOV (i.e. the spatial extent of the region accessible by the laser focal spot).

By far, the most widely used scanning mechanism for two-photon microscopy makes use of mirrors mounted on galvanometers. Mirrors have the advantage that they can handle high powers, provoke essentially no power loss, are equally efficient for a wide range of tilt angles, and are achromatic. However, the drawback of galvanometers is that they can be quite slow when used in a conventional raster-scanning mode, typically requiring about a second to perform an xy scan over the full microscope FOV. Several strategies have been adopted to improve image acquisition speed. For example, fast multi-photon imaging has been achieved by splitting the beam and scanning the sample with multiple beamlets (Bewersdorf et al., 1998, Kurtz et al., 2006). However, because two-photon microscopy is based on nonlinear excitation, the redistribution of laser power into multiple lower-power beamlets leads to a reduction in fluorescence power. Such a strategy also requires an imaging of fluorescence signal with an array detector, largely undermining the main advantage of two-photon microscopy when imaging in thick tissue (Helmchen and Denk, 2005).

Alternatively, fast single-beam scanning has been achieved with the use of resonant scanners (Fan et al., 1999) or rotating polygonal mirrors (Kim et al., 1999), though with the constraint of fixed raster-scan patterns. Recently, a spiral-scan strategy coupled with an oscillating focus has provided a remarkable 10 Hz scan rate over a 250 μm × 250 μm × 250 μm 3D volume (Göbel et al., 2007). Such strategies are highly effective for dense cell populations and indeed have been used to image hundreds of cells in a single scan. However, these strategies become less effective when the cells of interest are sparsely distributed because of their inefficient use of scan time: the laser beam spends little useful time illuminating the cells of interest while spending much dead time illuminating regions around these cells.

An alternative strategy to reduce dead time is the random-access approach where the laser focus is aimed only at specific cells of interest. This strategy has been adopted with two-photon microscopes based on acousto-optic deflectors (AOD's), the advantage being that AOD's are fast because they are inherently inertia-free (Iyer et al., 2006, Lechleiter et al., 2002, Roorda et al., 2004). However, AOD's impose their own constraints. Because their scan angles are much smaller than galvanometer's, they offer significantly reduced FOV's for a given microscope objective. Moreover, their transmission efficiencies are angle dependent and their chromatic dispersion leads to degraded resolution, which can only be partially corrected with complicated techniques of dispersion compensation (Lechleiter et al., 2002, Roorda et al., 2004, Salomé et al., 2006).

We provide the details here of a simple strategy, called targeted path scanning (TPS), which is an extension of a previously reported vector-mode approach (Nikolenko et al., 2007) that combines the advantages of mirror-based scanning and random-access targeting. Our strategy can readily be implemented using the hardware of standard two-photon microscopes, maintains a constant user-prescribed sampling rate and pixel size within cells of interest, and minimizes dead time between cells of interest. We demonstrate neuronal network activity measurements at 100 Hz scan rates over spatial ranges in the millimeter range, without sacrificing either the spatial resolution or the signal-to-noise ratio (SNR) associated with standard two-photon microscopy.

Section snippets

Electrophysiological recordings and staining

All protocols were approved by the Boston University Animal Care and Use Committee. Transverse hippocampal brain slices (400 μm) were prepared as previously described (Netoff et al., 2005) from Long-Evans rats aged P14–32. After a 1-h incubation period, they were transferred to the recording chamber. Slices were initially visualized using graded-field microscopy (Yi et al., 2006). The areas of interest were then stained with Calcium Green-1 AM (Invitrogen, Carlsbad, CA) using multicell bolus

Results

TPS relies on steering the laser beam along a well-defined path. To evaluate the speed at which the galvanometers can accurately track this path, we tested TPS with a worst-case-scenario scan path where three SOI's were separated by a large distance. The actual position of the galvanometer, provided by the galvanometer control electronics, was recorded for different values of maximal intercellular acceleration (i.e. for different scan rates). Although it was possible to intersect regions of

Discussion

We have described (and made available) a simple targeted-path strategy to constrain a laser scan path to user-defined SOI's and minimize dead time between these SOI's. Because the laser travels at a constant speed through each region of interest, a well-defined SNR is essentially guaranteed for regions of the same approximate size and stain concentration.

We note that our experimental demonstrations made use of the relatively high-affinity Ca2+-sensitive dye Calcium Green-1, which decays in

Acknowledgements

This work was partially funded by NIH grants EB005736, NS34425, MH61604, and the Burroughs Wellcome Fund 1001749.

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