The entrapment of squaraine (SQ) within a molecular container to form rotaxane has been shown to improve the dye stability and the fluorescence proficiency inside the mitochondria. The macrocycle provides shelter and protects the near-infrared (NIR) SQ chromophore from nucleophilic attacks made by the exposed thiol of Cys-containing mitochondrial proteins and mitochondrial glutathione. Herein a microwave-assisted template-directed clipping reaction on low-loading 2-chlorotrityl chloride resin is used to develop an NIR unsymmetrical squaraine rotaxane in high quantum yield.