3,3′-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy

Abstract

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3′-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and labmade mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2′-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

CAMSAP3 is required for mTORC1-dependent ependymal cell growth and lateral ventricle shaping in mouse brains

“…Stained sections were washed twice with PBS and mounted with either Fluorsave (Chemicon) or 2,2'-thiodiethanol (Harvard Center for Biological Imaging, hcbi.fas.harvard.edu/tips-and-tricks). For staining F-actin, phalloidin-CF488 (Biotium) was mixed with the secondary antibodies and sections were mounted with 3,3′-thiodipropanol (Tejedo et al, 2019). Whole-mount immunostaining was performed essentially in the same way as described above, except that no antigen retrieval was performed.…”

Super‐Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology

“…(2015); Musielak, Slane, Liebig, & Bayer (2016); Peterson, Mermelstein, & Meisel (2015); Tejedo et al. (2019).…”

Refractive-index matching enhanced polarization sensitive optical coherence tomography quantification in human brain tissue

“…In recent years, refractive index matching media, such as glycerol [10], 2,2'-thiodiethanol (TDE) [11], and 3,3'-thiodipropanol [12], have shown the capability to increase the imaging depth for fluorescence imaging [13,14]. The refractive index matching solution TDE can be miscible with water at any ratio and allows fine adjustment of the refractive index from that of water (1.33) to that of immersion oil (1.52).…”