Calcium imaging in temporal focus

Techniques for fast two photon laser scanning microscopy (2PLSM) of dynamic neural signals offer the prospect of minimally invasive functional mapping of neural circuitry. In a typical imaging scenario, a two-photon point spread function is raster scanned across a region of tissue, and pixels within regions of interest (e.g neuronal somata) are averaged to form a point in a time series. In galvanometric microscopes, this means that scan time is wasted in moving the point spread function around the cell, limiting signal acquisition rate. As a means of addressing this issue, we propose the use of temporally focused (TEFO) 2PLSM for monitoring dynamic signals. In contrast to the diffraction limited spot of 2PLSM, the TEFO spot is several orders of magnitude larger. It nonetheless preserves the axial confinement of the 2PLSM spot and also propagates such that multiphoton absorption is confined to the perifocal region. We demonstrate proof of principal by employing TEFO 2PLSM to image calcium signals in a thalamocortical slice stained with the calcium sensitive AM-ester dye (OGB-1 AM). We acquired Ca²⁺ signals in 2D line-scanning mode, at scan rates of 1 kHz, showing that action potential detection with relatively high temporal resolution can be achieved with this approach.