Asante Calcium Red Poster

Properties of Asante Calcium Red – a novel ratiometric indicator with long excitation wavelengths

Xenia A. Meshik*, Krzysztof L. Hyrc, Mark P. Goldberg**, Washington University School of Medicine, Dept. Neurology,

The Hope Center for Neurological Disorders, Alafi Neuroimaging Laboratory, Saint Louis, MO 63110.

ABSTRACT

Measurement of intracellular free calcium concentration ([Ca2+]i) is typically performed using fluorescent indicators. Ratiometric indicators (such as fura-2) allow calibrated [Ca2+]i measurement by comparison of two excitation or emission wavelengths. However, most ratiometric indicators require excitation with ultraviolet light, their emission wavelengths overlap with those of common fluorescent proteins such as GFP and YFP, and they are generally not suitable for use with standard confocal microscopes. In contrast, calcium indicators with visible excitation wavelengths (such as calcium-green) do not allow the accuracy of ratiometric measurements. We characterized the basic physicochemical properties of Asante Calcium Red (ACR), a newly available fluorescent indicator with long excitation and emission wavelength (TefLabs, Austin, TX).

First, we collected ACR excitation and emission spectra in standard calibration buffers to define the possible imaging modes and determine the indicator dissociation constants (Kd).  ACR can be excited with wavelengths ranging from 450 to 545 nm, with maximum emission at 640 nm, which allows imaging using the Argon laser available on standard confocal microscopes. The large Stokes’ shift allows using it as a single wavelength when excited with 453, 488 or 543 nm laser lines or as a ratiometric indicator by dividing the ACR emission at 640 and 530 nm when using 488 nm excitation. In either case, the indicator offers a large dynamic range as its emission at 640 nm increases about 30 times upon Ca2+ binding, whereas the isosbestic 530 nm emission is unaffected by Ca2+. Alternatively, the indicator might be imaged using 750-800 nm two-photon excitation in either single wavelength or ratiometric mode. The long emission wavelength permits [Ca2+]i determination in the presence of YFP or GFP. The dissociation constants for calcium were found to be 400 nM in the absence and 600 nM in the presence of 1 mM Mg2+. ACR fluorescence is sensitive to zinc (Kd=0.6 nM) and magnesium (Kd~20 mM). To examine the indicator behavior in situ, we loaded cultured neurons with ACR using a standard AM ester loading protocol (5 μM ACR/AM, 1 hour incubation) and imaged the cells using a Zeiss LSM510 META NLO multi-photon microscope. The emission and excitation spectra were similar to those in calibration buffers. The ACR fluorescence changed robustly in response to depolarization-induced calcium influx.

In summary, ACR combines long emission wavelengths and ratioing capabilities with very dynamic responses to changing [Ca2+]i.