Ultra-stable cryostage for cryo fluorescence microscopy applications

We present a sample holder to be used in cryo fluorescence microscopes or to simply and cost effectively upgrade a room temperature fluorescence microscope into a cryo fluorescence microscope. It has been shown that a clear correlation exists between temperature and photon yield on one side and photon yield and localization accuracy on the other side. Known problems of cryo temperature microscopes like temperature or mechanical instability are being solved by the design of the sample holder. A unique feature of the setup is the possibility to perform a sample change below the glass transition temperature of water. This makes it possible to study a vitrified sample correlatively by various techniques without destroying it through the formation of crystalline ice or ice condensation on the sample.

Challenges

2014's Nobel prize in chemistry has impressively shown that super-resolution fluorescence microscopy is an essential and increasingly important tool for studying biological systems. In fluorescence microscopy applications, biological structures are stained with fluorescent molecules and excited by laser irradiation of a particular wavelength during imaging. The colder the sample is, the longer the fluorescence molecules stay active, and, therefore, their localization accuracy increases. Controlling the thermal drifts and mechanical vibrations, both caused by the cooling system, are a major issue.