1：Experimental Design and Method Selection:

The study involved the design and synthesis of novel small-molecule fluorescently labeled probes for the in vitro imaging of KCa

2：1 channels. Senicapoc was selected as the targeting component due to its high affinity and selectivity towards KCa1 channels. Different BODIPY dyes were synthesized and connected to a propargyl ether derivative of senicapoc via a Cu-catalyzed azide alkyne [3+2]cycloaddition. Sample Selection and Data Sources:

The A549 cell line, a non-small-cell lung cancer (NSCLC) cell line, was used for all experiments due to its overexpression of the KCa

3：1 channel. List of Experimental Equipment and Materials:

The synthesis involved various chemical reagents and solvents, including tert-butyldiphenylsilyl chloride (TBDPS-Cl), n-butyllithium, 4-fluorobenzophenone, trimethylsilyl cyanide, InCl3, and propargyl bromide. Fluorescence microscopy was performed using an inverted fluorescence microscope.

4：Experimental Procedures and Operational Workflow:

The synthesis of the senicapoc derivative and BODIPY dyes was followed by their connection via a Cu(I)-catalyzed azide-alkyne [3+2]cycloaddition. The staining protocol involved incubating NSCLC cells with the fluorescently labeled ligands for 10 minutes, followed by washing with PBS-buffer.

5：Data Analysis Methods:

The photophysical properties of the BODIPY dyes were analyzed, including absorption and emission maxima, photoluminescence quantum yields, fluorescence lifetimes, and rate constants. The specificity of the probes was confirmed by pre-incubation with unlabeled senicapoc, and the density of KCa3.1 channels was compared between fluorescent probe staining and indirect immunofluorescence assay.