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Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impact cellular physiology would be facilitated by quantitative measurements of ER Ca2+ levels that allow for robust comparisons across conditions. To achieve this, we enhanced our original high dynamic range ER Ca2+ indicator, ER-GCaMP6-150, by fusing it to the HaloTag protein, which when bound to Janelia Fluor (JF) dyes creates a ratiometric ER Ca2+ probe. This probe (ER-Halo-GCaMP6-150) displayed minimal changes to the Ca2+-binding properties compared with our original ER Ca2+ probe as shown through in vitro and in cell Ca2+ calibrations. We describe a method to use this ratiometric probe for quantitative comparisons of ER Ca2+ concentrations and leverage this technique to compare ER Ca2+ levels across cell types and subcellular compartments. Using this approach, we show that the resting concentration of ER Ca2+ in primary dissociated neurons does not differ between excitatory and inhibitory subtypes nor between axonal and somatodendritic compartments. However, resting ER Ca2+ levels in neuronal somas are substantially lower than that measured in embryonic fibroblasts. The ER-Halo-GCaMP6-150 provides a robust tool to directly measure ER Ca2+ levels for studies of ER physiology across cell types and compartments.<\/jats:p>","DOI":"10.1085\/jgp.202413564","type":"journal-article","created":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T06:20:31Z","timestamp":1784960431000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":0,"title":["A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions"],"prefix":"10.1085","volume":"158","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4022-8707","authenticated-orcid":true,"given":"Ryan J.","family":"Farrell","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02r109517","id-type":"ROR","asserted-by":"publisher"}],"name":"Weill Cornell Medicine 1 Department of Biochemistry, , New York, NY,","place":["USA"]},{"name":"David Rockefeller Graduate Program, The Rockefeller University 2 , New York, NY,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3100-1914","authenticated-orcid":true,"given":"S. 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