Optimisation of fixation period on biological cells via time-lapse elasticity mapping

Chemical fixing is a standard approach for preserving the structural integrity of biological samples for downstream microscopy and analysis. However, the standard protocols are typically geared towards regular optical or fluorescence microscopy and this can lead to topographical artifacts during scanning probe microscopy imaging. Furthermore, there are many different fixatives and protocols available, and determining which is the most appropriate for a particular cell line (that will subsequently require imaging by scanning probe microscopy) is often important. The rationale for this study was to review fixation approaches in the context of probe microscopy and to determine whether elasticity mapping is a useful tool for estimating the optimal fixing duration. Our findings suggest that, for the human breast carcinoma cell line MCF7 fixed in 4% fresh buffered formaldehyde, the degree of cross linking approaches completeness within 30 min. Knowledge of this is important, particularly in the context of the adjunct technique of fluorescence microscopy as under fixation causes the loss of signals and poor preservation of morphological detail, whereas over fixation causes the loss of signals and increases background noise from non specific signals. Thus, such measurements represent the best compromise for fixation approaches in preparation for tandem scanning probe and fluorescence/confocal studies.