3D visualization of the intact vasculature is crucial for understanding the pathogenesis of different neurological and vascular diseases. Although various fluorescent vessel labeling methods have been already used for overall visualization of different vascular networks, little has been done to quantify the labeling performance of each vessel labeling method, as well as their applicability with various clearing protocols. In this study, we systemically compare the labeling performance of four vessel labeling methods on different mouse organs. We also quantitatively evaluate the compatibilities of the four vessel labeling methods with several typical tissue optical clearing methods. Finally, we select the optimal combination of tested vessel labeling and clearing method to perform 3D reconstruction of vascular networks within several typical organs. This study is expected to provide important information for choosing proper pipeline for 3D visualization of different vascular networks.
SignificanceVisualization of intact vasculatures is crucial to understanding the pathogeneses of different neurological and vascular diseases. Although various fluorescent vessel labeling methods have been used in combination with tissue clearing for three-dimensional (3D) visualization of different vascular networks, little has been done to quantify the labeling effect of each vessel labeling routine, as well as their applicability alongside various clearing protocols, making it difficult to select an optimal combination for finely constructing different vasculatures. Therefore, it is necessary to systematically assess the overall performance of these common vessel labeling methods combined with different tissue-clearing protocols.AimA comprehensive evaluation of the labeling quality of various vessel labeling routines in different organs, as well as their applicability alongside various clearing protocols, were performed to find the optimal combinations for 3D reconstruction of vascular networks with high quality.ApproachFour commonly-used vessel labeling techniques and six typical tissue optical clearing approaches were selected as candidates for the systematic evaluation.ResultsThe vessel labeling efficiency, vessel labeling patterns, and compatibility of each vessel labeling method with different tissue-clearing protocols were quantitatively evaluated and compared. Based on the comprehensive evaluation results, the optimal combinations were selected for 3D reconstructions of vascular networks in several organs, including mouse brain, liver, and kidney.ConclusionsThis study provides valuable insight on selecting the proper pipelines for 3D visualization of vascular networks, which may facilitate understanding of the underlying mechanisms of various neurovascular diseases.
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 (RT). Therefore, it is important to solve the contradiction between fluorescence preservation and imaging condition. We develop a modified FDISCO clearing method, termed FDISCO+, to change the preservation condition from low temperature (LT) to RT. Two alternative antioxidants were screened out to effectively inhibit the peroxide generation in the clearing agent at RT, enabling robust fluorescence preservation of cleared samples. FDISCO+ achieves comparable fluorescence preservation with the original FDISCO protocol and allows long-time storage at RT, making it easier for researchers to image and preserve the samples. FDISCO+ is expected to be widely used due to its loose operation requirements.
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.
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