Chemical calcium indicators

Abstract

Our understanding of the underlying mechanisms of Ca2+ signaling as well as our appreciation for its ubiquitous role in cellular processes has been rapidly advanced, in large part, due to the development of fluorescent Ca2+ indicators. In this chapter, we discuss some of the most common chemical Ca2+ indicators that are widely used for the investigation of intracellular Ca2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators now available allow for intracellular Ca2+ detection over a very large range (<50 nM to >50 microM). High affinity indicators can be used to quantify Ca2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca2+ detection when more than one fluorophore is being imaged. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca2+ indicators including uneven dye loading, leakage, photobleaching, and changes in cell volume. Recent technical advances that permit in vivo Ca2+ measurements will also be discussed.

Figure 1. Optical Imaging in vivo of the mouse parietal cortex

Left panel shows the objective inverter attached to a Zeiss LSM 510 multiphoton microscope positioning the 60x 1.1 NA water immersion objective above the mouse parietal cortex. Right panel is a higher magnification of the stainless steel ring holder that is glued to the skull and immobilizes the brain. The center ring has been filled with 2 % agarose (Sigma type VII) and sealed from above with a glass coverslip (#0). This essentially eliminates motion artifacts due to breathing when the hole in the cranium is less than 1-2 mm in diameter. The red heating pad is maintained at 37°C.

Figure 2. In vivo Ca²⁺ imaging in mouse cortical astrocytes using Fluo-4 AM

A mouse was anesthetized with isoflurane and the cortex prepared as described in the text. Fluo-4 AM (50 μg) was vortexed with 5 μl Pluronic F-127, mixed with ASF to a final concentration of 100 μM and pipetted onto the cortical surface for ~60 minutes. (A) In vivo images of the mouse cortex loaded with Fluo-4 AM at the three time points as labeled. Images were collected on a Nikon C1si confocal microscope fitted with an objective inverter with a 40X objective. A time course of Fluo-4 fluorescence was collected with images acquired every 5 seconds. Resting levels of Ca²⁺ were imaged (left panel) before the P2Y1R agonist, 2MeS ADP (100μM), was added to the cortex. The middle panel shows the peak Fluo-4 fluorescence in response to 2MeS ADP. The right panel shows the recovery of the cells from 2MeS ADP (380 s). (B) Lineplot of the averaged fluorescence intensity (F) of the five individual cells identified in the right panel of (A). (C) Graph of the same data in (B), but plotted as ΔF/F using the formula (F-F_rest)/F_rest. Note the smaller standard error compared to that in (B). Data was analyzed using Image J.