FDISCO+: a clearing method for robust fluorescence preservation of cleared samples.

Significance: The recently reported solvent-based optical clearing method FDISCO can preserve various fluorescent signals very well. However, the strict low-temperature storage condition of FDISCO is not conducive to long-time or repetitive imaging usually conducted at room temperature. Therefore, it is important to solve the contradiction between fluorescence preservation and imaging condition. Aim: We develop a modified FDISCO clearing method, termed FDISCO+, to change the preservation condition from low temperature to room temperature. Approach: Two alternative antioxidants were screened out to effectively inhibit the peroxide generation in the clearing agent at room temperature, enabling robust fluorescence preservation of cleared samples. Results: FDISCO+ achieves comparable fluorescence preservation with the original FDISCO protocol and allows long-time storage at room temperature, making it easier for researchers to image and preserve the samples. Conclusions: FDISCO+ is expected to be widely used due to its loose operation requirements.