Measuring Ca2+ Dynamics in the Astrocytic Syncytium with FLIM: Side-by-Side Comparison of Bolus Versus Patch Loading of Ca2+-Sensitive Dyes

Chemical dyes have been widely used in calcium (Ca2+) imaging to study cell physiology in the brain. However, the dye introduction method can complicate the interpretation of results. In this study, we compare two techniques of dye loading into astrocytes: bolus loading of AM dyes and patch loading. While bolus loading is less invasive, it mainly targets cell bodies and may not accurately capture events in fine astrocytic processes, leading to potential false negatives. On the other hand, patch loading is more effective in labeling processes, but it may cause mechanical disturbances that could distort the results. To navigate these complexities, we performed a comparison analysis and a methodology using fluorescence lifetime imaging (FLIM) for monitoring intracellular calcium ion concentrations ([Ca2+]) in astrocytic somata and protrusions within acute brain slices. The methodology includes selecting a suitable Ca2+-sensitive fluorescent dye and its calibration for [Ca2+] readout using OGB-1 as an example. We compared the effects of bolus-loading OGB-1 AM dye against whole-cell patch-loading cell-impermeable OGB-1 on astrocyte Ca2+ and showed how these dye delivery methods may differentially report the effects of G-protein-coupled estrogen receptor activation on astrocyte Ca2+ signaling.