2,2′-Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy

Autor: Rebecca Medda, Johann Engelhardt, Marion C. Lang, Stefan W. Hell, Thorsten Staudt
Rok vydání: 2006
Předmět:
Zdroj: Microscopy Research and Technique. 70:1-9
ISSN: 1097-0029
1059-910X
DOI: 10.1002/jemt.20396
Popis: The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging10 micro m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.
Databáze: OpenAIRE
Externí odkaz: