Laser scanning microscopy is emerging as a powerful imaging tool in cryobiology. The
basic microscopy system can be combined with various imaging modalities including
Raman spectroscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging,
or multiphoton imaging. Multiphoton imaging can be used to study intracellular ice
formation at the subcellular level. A Raman imaging modality can be used for chemical
mapping of frozen samples. A Raman spectrum gives information about characteristic
molecular vibrations of specific groups in molecules. Raman images can be used to
determine the localization of intra-and extracellular constituents and the various
forms of water in freeze-concentrated solutions. Spectra can be collected during freezing
and thawing of a sample using a temperature-controlled sample holder. In this chapter,
various advanced cryoimaging methods are described. Special emphasis is given on the
different imaging modalities that can be used to study the various aspects of cryopreservation.
