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Here you can find the parts lists, assembly instructions and other documentation of the "scanned temporal focusing" - Scan-TeFo microscope for optimized 2-photon calcium imaging.

Introduction

In this work, we present a novel method based on light sculpting that enables unbiased single and dual-plane high-speed (up to 160 Hz) calcium imaging, as well as in vivo volumetric calcium imaging of a mouse cortical column (500x500x500 µm) at single-cell resolution and fast volume rates (3 - 6 Hz). This is achieved by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio while using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. Together, these innovations have enabled the near-simultaneous in-vivo recording of calcium dynamics of several thousand active neurons across cortical layers and in the hippocampus of awake behaving mice.

In our approach, termed scanned temporal focusing (s-TeFo), we have synergistically combined three key ideas; (1) to excite the minimally required number of voxels per unit volume necessary to resolve the structure of interest, (2) to ensure a near-isotropic 3D shape of these excitation voxels and (3) to maximize the obtainable fluorescence signal-to-noise from each voxel by synchronizing the laser pulse repetition to the voxel acquisition rate. These conditions increase the volume acquisition rate by eliminating the unnecessary oversampling in the lateral (x,y) plane as is some of the applications of conventional 2PM just for obtaining the necessary axial localization of excitation. At the same time, it allows maximizing the obtainable fluorescence signal-to-noise for a given average excitation power. The enlarged sculpted PSF volume in combination with the one pulse per voxel excitation scheme allows for a higher total signal-to-noise ratio from each GCaMP-expressing neuron at the same excitation power density. This is in essence because larger voxels lead to a higher total collected fluorescence from each voxel and also because at a lower total number of voxels a lower repetition rate can be used – while maintaining a one-pulse per pixel excitation condition – from which the signal benefits non-linearly. Thus, for the same obtainable signal to noise ratio we can either reduce the laser pulse energy per unit area and time (J/(µm2/s)) which in turn improves cell viability or the higher obtainable signal-to-noise ratio in case of s-TeFo can be used to reduce the voxel dwell time and thereby speed up the voxel acquisition rate. Furthermore, as shown previously, using temporal focusing also provides the additional advantage that the axial confinement of excitation is more resilient to aberrations and tissue scattering compared to conventional two-photon excitation.

Figure 1: Schematic and principle of scanned temporal focusing imaging system. (a) Schematic of the s-TeFo imaging. A large field-of-view is raster-scanned using an enlarged sculpted PSF and a one-pulse-per voxel excitation-acquisition scheme. Volumetric image acquisition is achieved by translating the objective axially (along z-axis) via a high-speed long-range piezo. Scale bar is 100µm. (b) Measured axial confinement of the sculpted point-spread-function (PSF) of the laterally symmetric TeFo-spot using 0.5µm sized beads. Corresponding x and z profiles showing lateral and axial confinement of excitation, a.u., arbitrary units. Scale bar is 10µm. (c) Overview of the s-TeFo microscope. The main dichroic mirror and a mirror are mounted on a slide in order to switch between two-photon scanning and wide-field epi-fluorescent imaging mode. EOM, electro-optical modulator, BS, beam-splitter, PBS, polarizing beam-splitter.

A schematic of our s-TeFo imaging approach is depicted in Fig. 1a along with a measurement of the corresponding 3D PSF (Fig. 1b), and the schematic of the setup (Fig. 1c). The enlarged 3D PSF has about a 130-fold increased excitation volume of ~130 µm3 compared to the PSF in diffraction-limited scanning. This required a corresponding increase in pulse energy in order to maintain a comparable power density (J/(µm2/s)), which in turn necessitated the development of an alternative femtosecond laser that can provide sufficient pulse energies at repetition rates equal to or higher than the image acquisition rate. Whereas a repetition rate higher than the imaging/voxel rate would be possible in principle, the highest signal-to-noise is achieved with a one-pulse-per-voxel excitation approach, as this scheme optimizes the obtainable signal and prevents integration of detector read noise that can accumulate when fluorescence from multiple pulses is integrated.

Relevant publications

• Robert Prevedel, Aart J. Verhoef, Alejandro J. Pernia-Andrade*, Siegfried Weisenburger*, Ben S. Huang, Tobias Nöbauer, Alma Fernández, Jeroen E. Delcour, Peyman Golshani, Andrius Baltuska and Alipasha Vaziri, "Fast volumetric calcium imaging across multiple cortical layers using sculpted light", Nature Methods, in press (2016).

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