An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging

Although live cell imaging is desirable, it is not always feasible and in many situations cells are fixed in order to provide a ‘snapshot’ of the nature and distribution of molecules within a cell while minimising changes from cell movement, sample degradation etc. There is a wide range of fixation methods available that act via different mechanisms, and on different cell components. Each method has advantages and disadvantages and a choice of what fixation method to choose for a particular experiment needs to take these factors into consideration. Here we used Raman spectroscopic imaging of live cells, and compared with cells preserved with aldehyde, or organic solvent-based fixation methods to assess the chemical changes induced by each fixative, and their impact on the quality of images that can be obtained from fixed cells. Overall, aldehyde fixation methods performed significantly better than organic solvents with less severe loss of biochemical information. Aldehyde based fixatives show an altered biochemical content of the cells, attributed to adduct formation, but this can be minimised by optimising fixation temperature, or through removal of adduct formation by detergent-based permeabilization treatments as a second step (at the cost of the loss of other biochemical information). The results showed that organic solvents, on the other hand, lead to a severe loss of cell content, attributed to the loss of membrane integrity after the removal of lipids. Additionally, fixation with aldehydes prior to permeabilization with organic solvents does not provide adequate protection of cytoplasmic content. The use of Raman imaging is ideal for comparing groups of cells in terms of their molecular content, and the results show that aldehyde fixations methods are preferable for studies where the overall molecular content of the samples is important. Although there is no universal fixation method for every application, the results here allow us to provide a few general principles: where spectral similarity to live cells is important, fixation with paraformaldehyde at room temperature is preferable, at the cost of some blebbing and vacuole formation. Where preservation of cellular structure or biomolecular distribution is important, a mix of paraformaldehyde and glutaraldehyde would be more appropriate, but at the cost of some changes to spectral profile, particularly in DNA-related bands.