Microscopic Imaging of Intracellular Calcium in Live Cells Using Lifetime-Based Ratiometric Measurements of Oregon Green BAPTA-1

Abstract

Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer's disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantages to using a ratiometric approach, which allows quantitative determinations of calcium that are independent of dye concentration. Taking advantage of the distinct lifetime contrast of the calcium-free and calcium-bound forms of OGB, we used time-domain lifetime measurements to generate calibration curves for OGB lifetime ratios as a function of calcium concentration. In summary, we demonstrate approaches using commercially available tools to measure calcium concentrations in live cells at multiple scales using lifetime contrast. These approaches are broadly applicable to other fluorescent readouts that exhibit lifetime contrast and serve as powerful alternatives to spectral or intensity readouts in multiplexing experiments.

Astrocytic endfoot Ca2+ correlates with parenchymal vessel responses during 4-AP induced epilepsy: An in vivo two-photon lifetime microscopy study

“…The lifetime decay curves for OGB-1 in each of 10 Ca 2+ buffers were measured using samples of free dye in glass capillary tubes at varying Ca 2+ concentrations. 26 In the absence of Ca 2+ , OGB-1 had a single, fast decay leading to an effective lifetime of ∼0.65 ns accounting for the PMT response function. At saturating levels of Ca 2+ (1.35 µM), OGB-1 had a single, slow decay leading to a lifetime of ∼4.86 ns.…”

Municipal bylaw to reduce cosmetic/non-essential pesticide use on household lawns - a policy implementation evaluation

“…TPH incorporated information from an environmental scan of initiatives in other jurisdictions into a policy document [31] with four options: A) public education only, as carried out in Seattle, King County [32]; B) voluntary compliance approach, as in most recycling programs; C) bylaw (all properties), such as Oregon state's Pesticide Tracking Law, also called a "right-to-know" Law [33] and D) bylaw (vulnerable populations only) as in Washington State's children's law around notification of pesticide spraying in schools [34] or New York's Neighbor Notification Law [35]. …”

Luminescence lifetime imaging of three-dimensional biological objects

“…(1) Reviewed in Looger and Griesbeck ( 2012 (3) Ca 2+ -sensing dye Oregon Green BAPTA-1 (Kuchibhotla et al, 2009;Lattarulo et al, 2011). Requires commercially available confocal or two-photon FLIM.…”

“…Additional parameters that provide information on the function of excitable cells can also be sensed using FLIM. For example, FLIM can be used to monitor intracellular Ca 2+ concentration via genetically encoded Ca 2+ indicator (GECI) biosensors (Looger and Griesbeck, 2012;Tian et al, 2012) and small-molecule probes such as Ca 2+ -chelating Oregon Green BAPTA-1 (Kuchibhotla et al, 2009;Lattarulo et al, 2011) (Table 1). Traditionally, FLIM has been considered to be too slow for capturing fast Ca 2+ changes.…”