Infrared microscopy for in situ measurement of protein secondary structure during freezing and freeze-drying

A commercially available freeze-dry microscopy stage interfaced with an IR microscope is described as a method of in situ measurement of protein secondary structure in the liquid, frozen and freeze-dried states. Studies using solutions of model proteins demonstrated that spectra collected using the IR microscope have resolution and sensitivity that is comparable to techniques using a conventional infrared spectrometer. Additionally, spectra collected in triplicate on the microscope in the solution, frozen, and freeze-dried states and after reconstitution were shown to be reproducible. The limiting factor when collecting spectra on the infrared microscope appears to be the higher level of water vapor inherently present within the optical path of the microscope used in this study. Results demonstrate that the native secondary structure is perturbed in both the frozen and freeze-dried states, and bands characteristic of structural changes associated with freezing and drying stresses were observed in the Amide I region. Freeze-drying studies conducted in the presence of mannitol and sucrose demonstrated that perturbation to the native state secondary structure after freeze-drying was considerably reduced in the presence of these excipients.