TY - JOUR TI - Sources of widefield fluorescence from the brain AU - Waters, Jack A2 - Yasuda, Ryohei A2 - Huguenard, John R VL - 9 PY - 2020 DA - 2020/11/06 SP - e59841 C1 - eLife 2020;9:e59841 DO - 10.7554/eLife.59841 UR - https://doi.org/10.7554/eLife.59841 AB - Widefield fluorescence microscopy is used to monitor the spiking of populations of neurons in the brain. Widefield fluorescence can originate from indicator molecules at all depths in cortex and the relative contributions from somata, dendrites, and axons are often unknown. Here, I simulate widefield illumination and fluorescence collection and determine the main sources of fluorescence for several GCaMP mouse lines. Scattering strongly affects illumination and collection. One consequence is that illumination intensity is greatest ~300–400 µm below the pia, not at the brain surface. Another is that fluorescence from a source deep in cortex may extend across a diameter of 3–4 mm at the brain surface, severely limiting lateral resolution. In many mouse lines, the volume of tissue contributing to fluorescence extends through the full depth of cortex and fluorescence at most surface locations is a weighted average across multiple cortical columns and often more than one cortical area. KW - widefield KW - fluorescence KW - imaging KW - microscopy KW - cortex JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -