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

Milvia Iris Alata Tejedo,Juan Carlos Martínez Cervantes,Valeria Piazza,Arturo González Vega,Adrian Saul Jimenez Roldán,Mario Rodriguez

doi:10.1364/boe.10.001136

Milvia Iris Alata Tejedo, Juan Carlos Martínez Cervantes + Show 4 more

Open Access

https://doi.org/10.1364/boe.10.001136

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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 lab-made 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.

Highlights

Use of high numerical aperture lenses, low background staining protocols, high quality glass for slide preparation are common steps that can be undertaken to improve the quality of the fluorescence images that could be generated from biological microscopy slides
The experimental set of data obtained corresponds to nD and is shown in Fig. 1, where the linear relation found between refractive index and concentration in water is reported
TDPs optical characteristics are suitable for high resolution fluorescence microscopy of multi-stained samples, with the additional benefit of being compatible with the most common molecule used for labeling filamentous actin in fixed cells, phalloidin [10]

Summary

Introduction

Use of high numerical aperture lenses, low background staining protocols, high quality glass for slide preparation are common steps that can be undertaken to improve the quality of the fluorescence images that could be generated from biological microscopy slides Another aspect that needs to be considered in the staining and mounting protocol is the chemical substance that is used to seal the sample between the slide and the coverslip. In addition to the lack of adverse effect on tissue components, it is desirable for the mounting medium to be harmless for the user Another often disregarded characteristic is the refractive index (n) of the mounting medium, which for high resolution, diffraction-limited microscopy should be as close as possible to that of glass and immersion oil, i.e., 1.515 (by convention measured at the sodium spectral line). The maximal homogeneity in n at the two coverslip boundaries is required to avoid size scaling in the z axis, reduction of the effective numerical aperture and reduction in resolution and peak intensity [1]

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