Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence measurements from 34-91 layer 2/3 neurons at a 180-490 Hz sampling rate. We detected single action potential-evoked calcium transients with signal-to-noise ratios of 2-5 and determined spike times with near-millisecond precision and 5-15 ms confidence intervals. An automated 'peeling' algorithm enabled reconstruction of complex spike trains from fluorescence traces up to 20-30 Hz frequency, uncovering spatiotemporal trial-to-trial variability of sensory responses in barrel cortex and visual cortex. By revealing spike sequences in neuronal populations on a fast time scale, high-speed calcium imaging will facilitate optical studies of information processing in brain microcircuits.

Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community.

Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes.