Cryo-fluorescence microscopy

Low-temperature microscopy enables biological observations to be made under near-natural conditions. Analyses of biological samples in ambient conditions often have to be made at the cost of restricting the vitality of the sample. This is typically achieved by fixing the sample chemically, which in turn often causes structural and chemical alterations. In contrast to this, when samples are flash frozen for low temperature microscopy, their original structures are preserved almost entirely, and chemical changes are minimal. Such rapid freezing techniques are known in electron or x-ray or low temperature microscopy, for example (Hurbain und Sachse, 2011; Schneider et al., 2012).

Other advantages of using low temperature fluorescence microscopy to measure samples include:

  • Stabilisation of the sample against bleaching (Schwartz et al., 2007)
  • Increase of the fluorescence yield (Schwartz et al., 2007)
    • when measuring fluorescing proteins
    • when using fluorescence markers at lower concentrations
    • with substance-specific auto-fluorescences
  • Improvement of the signal / noise ratio (Giske 2007)
  • Narrowing of excitation bands
  • Stabilisation of the sample against infiltration by higher-energy radiation (UV)

Giske A. University of Heidelberg; 2007. CryoSTED microscopy — a new spectroscopic approach for improving the resolution of STED microscopy using low temperature. (PhD thesis)
Hurbain I., Sachse M. The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol Cell. 2011;103:405–420.

Schneider G., Guttmann P., Rehbein S., Werner S., Follath R. Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging. J Struct Biol. 2012;177:212–223.

Schwartz C.L., Sarbash V.I., Ataullakhanov F.I., McIntosh J.R., Nicastro D. Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc. 2007;227:98–109.