Intracellular calcium can be monitored by various calcium-specific fluorescent dyes including gene-encoded calcium indicators (GECI). GCaMP is a widely-used GECI that emits green fluorescence proportional to the level of intracellular calcium. However, since many tagging proteins also emit green fluorescence, GCaMP cannot be used with another green fluorescent protein. Therefore, it would be ideal to develop a GECI that has a distinct color profile other than green. In this regard, we developed a novel blue fluorescent calcium indicator modified from GCaMP called Ser²²²-Ala²²⁹-Cys³³⁰-BCaMP3. Specifically, a simple threonine to histidine substitution to a green fluorescent Cys³³⁰-GCaMP3 successfully changed its fluorescence to blue (Cys³³⁰-BCaMP3, B for blue). Furthermore, a couple of additional amino acid substitutions resulted in more enhanced blue fluorescence intensity. Among other Cys³³⁰-BCaMP3 variants, it was found that Ser²²²-Ala²²⁹-Cys³³⁰-BCaMP3 exhibited the strongest blue fluorescence intensity. When Ser²²²-Ala²²⁹-Cys³³⁰-BCaMP3 was co-expressed with TRPA1 – a non-selective cation channel – in HEK293T cells, it showed moderate blue fluorescence. One of the drawbacks of Ser²²²-Ala²²⁹-Cys³³⁰-BCaMP3 was that the fluorescence intensity was not enough when cells were cultured under 37°C. However, this limitation was circumvented by lowering cell culture temperature to 28°C, allowing much more enhanced blue fluorescence. Although Ser²²²-Ala²²⁹-Cys³³⁰-BCaMP3 mandates further optimization, the present study has found a promising blue fluorescent GECI that is derived from GCaMP3.

胞内钙可以通过多种钙特异性荧光染料进行监测，包括基因编码的钙指示剂（GECI）。GCaMP是一种广泛使用的GECI，它发出与细胞内钙水平成比例的绿色荧光。但是，由于许多标记蛋白也发出绿色荧光，因此GCaMP不能与其他绿色荧光蛋白一起使用。因此，理想的是开发一种具有不同于绿色的颜色配置文件的GECI。在这方面，我们开发了一种由GCaMP改性的新型蓝色荧光钙指示剂，称为Ser ²²² -Ala ²²⁹ -Cys ³³⁰ -BCaMP3。具体来说，简单的苏氨酸到组氨酸的取代就变成了绿色荧光Cys ³³⁰-GCaMP3成功地将其荧光更改为蓝色（Cys ³³⁰ -BCaMP3，B表示蓝色）。此外，另外两个氨基酸取代导致蓝色荧光强度增强。在其他Cys ³³⁰ -BCaMP3变体中，发现Ser ²²² -Ala ²²⁹ -Cys ³³⁰ -BCaMP3表现出最强的蓝色荧光强度。在HEK293T细胞中，当Ser ²²² -Ala ²²⁹ -Cys ³³⁰ -BCaMP3与TRPA1（非选择性阳离子通道）共表达时，它显示出中等的蓝色荧光。Ser ²²² -Ala ²²⁹ -Cys ³³⁰的缺点之一-BCaMP3是当在37℃下培养细胞时荧光强度不足。但是，通过将细胞培养温度降低到28°C可以避免这种限制，从而可以大大增强蓝色荧光。尽管Ser ²²² -Ala ²²⁹ -Cys ³³⁰ -BCaMP3要求进一步优化，但本研究发现了有前途的蓝色荧光GECI，其源自GCaMP3。