Particle-Based Optical Sensing of Intracellular Ions at the Example of Calcium - What Are the Experimental Pitfalls?

Abstract

Colloidal particles with fluorescence read-out are commonly used as sensors for the quantitative determination of ions. Calcium, for example, is a biologically highly relevant ion in signaling, and thus knowledge of its spatio-temporal distribution inside cells would offer important experimental data. However, the use of particle-based intracellular sensors for ion detection is not straightforward. Important associated problems involve delivery and intracellular location of particle-based fluorophores, crosstalk of the fluorescence read-out with pH, and spectral overlap of the emission spectra of different fluorophores. These potential problems are outlined and discussed here with selected experimental examples. Potential solutions are discussed and form a guideline for particle-based intracellular imaging of ions.

Intracellular delivery and sensing based on polyelectrolyte multilayer capsules

“…the sensing of two or more analytes simultaneously, is limited due to spectral overlap of the dyes [42]. Additionally one has to keep in mind when working with dyes, that crosstalk can occur from different ions or pH [39,43]. As will be highlighted further in the section on sensing, many organic fluorophores are sensitive to more than one ion and the fluorescence may also be dependent on the local pH.…”

“…However, many of the dyes available tend to be pH-sensitive in addition to respond to their specific ion and may also be sensitive to other ions [82]. For example, the potassium-and sodium-sensitive dyes, a fluorescent potassium-sensitive indicator (PBFI) and a fluorescent sodium-sensitive indicator (SBFI), respectively, show crosstalk with the correspondent other ion [39,43], and the Mg 2+ -sensitive dye MagIndo-1 can also be used for sensing Ca 2+ . In Figure 10, the pH dependence of the chloride-sensitive dyes: N-(ethoxycarbonylmethyl])-6-methoxy--quinolinium bromide (MQAE) and 10,10'-bis(3-carboxypropyl)-9,9'--acridiniumdinitrate (BAC), the calcium sensor dye Calcium Green-1, and the magnesium sensor dye Mag-Indo-1 are reported by Kantner et al [43].…”

“…For example, the potassium-and sodium-sensitive dyes, a fluorescent potassium-sensitive indicator (PBFI) and a fluorescent sodium-sensitive indicator (SBFI), respectively, show crosstalk with the correspondent other ion [39,43], and the Mg 2+ -sensitive dye MagIndo-1 can also be used for sensing Ca 2+ . In Figure 10, the pH dependence of the chloride-sensitive dyes: N-(ethoxycarbonylmethyl])-6-methoxy--quinolinium bromide (MQAE) and 10,10'-bis(3-carboxypropyl)-9,9'--acridiniumdinitrate (BAC), the calcium sensor dye Calcium Green-1, and the magnesium sensor dye Mag-Indo-1 are reported by Kantner et al [43]. Fluorescence signal of the chloride-sensitive dyes (a) MQAE and (b) BAC, in dependence of Cl -and pH, (c) calcium-sensitive Calcium-green-1 against Ca 2+ and pH, and (d) magnesium-sensitive Mag-Indo-1 in dependence of Mg 2+ and pH.…”

“…In all cases, the signal intensity is clearly dependent on pH in addition to the respective ion. The image is taken from reference [43]. Reprint with permission from Wiley-VCH.…”

“…There are numerous publications on optical sensors for intracellular ion sensing. However, very often there are also numerous shortcomings included [43,92]. In many articles, sensor systems are presented that show very good behavior in defined environment, for example buffers with varying concentration of the targeted ion.…”