Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates

Abstract

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.

Fig. 1.

(A) Schematic of the fast focusing two-photon imaging system. x-y scanning was carried out by two galvanometers in the lateral scan unit (LSU) and z-scanning by the mirror M in the axial scan unit (ASU). A polarizing beam splitter (PBS) and quarter-wave plate (QWP) directed all light through the ASU and into the imaging objective (L₂). (B) Theoretical wavefronts and intensity distributions produced for various settings of the LSU and ASU. In the central position, where mirror M lies in the focal plane of L₁, the resulting wavefront is flat. For positive and negative displacements of the mirror, curved wavefronts are produced and the spot refocuses along the axis. The effect of the LSU is to tilt the wavefronts, which displaces the spot laterally in the specimen and the combination of both ASU and LSU together can shift the spot to any 3-dimensional location in the specimen.

Fig. 2.

Ray diagrams showing the ray paths in the focal regions of L₁ and L₂ for two different focal settings. In each case all rays focus through a single point in the specimen irrespective of the focal setting.

Fig. 3.

(A) Custom-built actuator for scanning the mirror M in the ASU. The mirror M is mounted on a flexible beryllium copper bridge and two further galvanometers, G1 and G2, rotate synchronously to produce axial motion. (B) The experimentally measured frequency response curve for this actuator when scanning the focal spot 25 μm along z direction in the specimen.

Fig. 4.

Non-normalized experimental measurements of the focal spot intensity distribution in the x-z plane for different focal settings of the microscope with corresponding peak intensities labeled in arbitrary units. Scale bar 2 μm.

Fig. 5.

Experimental verification of arbitrary line scanning. Black squares mark points of interest through which it is desired to scan the focal spot. A continuous trajectory is defined by fitting periodic cubic splines through these and a correction algorithm is applied to compensate for the response characteristics of the galvanometers. Dashed blue and solid red lines indicate respectively the trajectory followed before and after correction. (A) Scanning two points at a sampling rate of 1 kHz. (B) Scanning points on a three-dimensional Lissajous figure with a sampling rate of 500 Hz. (C) Scanning points on a complex arbitrary trajectory at 300 Hz. After correction, the focal spot passes within 0.5 μm of each desired point in all three trajectories.

Fig. 6.

Functional imaging of cortical neurons. (A) Imaging was performed in cortical slabs to emulate the use of this method in the in vivo setting. (B) Projection of three-dimensional image stack in x-z plane showing a single cell loaded with Fluo-4 and Alexa 594. (C) Three-dimensional rendering of a single cell with two points marked that are separated by more than 30 μm in the z direction. (D) Fluorescence measurements from points of interest taken with a temporal resolution of 500 Hz. (E) Three-dimensional rendering of neuronal population bolus-loaded with OGB1-AM. (F) Three-dimensional representation of the neurons in the scanned volume (100 × 100 × 100 μm). (G) Fluorescence measurements from different points in the stack in response to extracellular electrical burst stimulation taken with a temporal resolution of 1 kHz and over more than 60 μm z distance. Traces represent the average of 2–4 trials.