| Description |
Genetically encoded calcium indicators (GECIs) are Ca2+ sensitive fluorescent proteins that have expanded the usefulness of optical calcium imaging to longitudinal in vivo studies due to their advantage of direct expression in the tissue being imaged. Several generations of GECIs have been developed using green fluorescent protein (GFP) or one of its variants with each generation improving upon Ca2+-binding affinities and optical properties. However, the tissue penetration of excitation or emission light through tissue is small due to high absorption of available GECI wavelengths, which are shorter than the infrared range. The field still lacks a GECI with excitation or emission wavelengths in the infrared range, which has significantly less attenuation in biological tissue. Here we propose the development of an infrared GECI by insertion of the Ca2+-binding domain calmodulin (CaM) into regions surrounding the biliverdin chromophore binding pocket of infrared fluorescent protein (iRFP). We proposed seven DNA constructs of iRFP with different CaM insertion sites. Six of the seven DNA constructs were successfully produced with protein expressed from one of these constructs exhibiting similar optical properties to iRFP, showing successful receptor insertion into iRFP. Though our initial Ca2+ sensitivity test to monitor change in fluorescence due to Ca2+ binding is not conclusive, we open the field of GECI engineering to exciting new possibilities for noninvasive deep tissue calcium imaging. |
