The Reliability of Fluorescence Z-Scan Analysis in the Complex Environment of the Living Cell

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Fluorescence z-scan analysis aims to fit the intensity traces recorded while moving a two-photon excitation volume vertically through a cell in order to identify the vertical concentration profile of a fluorescent species. Z-scan analysis has proved able to quantify delicate PM-protein binding interactions as well as sub-resolution partitioning of proteins due to the actin cortex. Despite recent progress, questions remain regarding the reliability and applicability of z-scan analysis in the complex environment of the living cell. In high precision z-scan applications, obtaining a good quality of fit is critical to ensuring that experimental results remain uncontaminated by fluorescent features outside the scope of the z-scan modeling. However, a statistical method to robustly assess the quality of fit in z-scan analysis has been lacking. To address these issues, we provide control data validating core aspects of the z-scan method at high precision and demonstrate the potential for error when applying the method without rigorous quality of fit controls. We propose a conceptual framework for estimating the amplitude of errors in z-scan analysis due to fluorescent features that may not be included in the z-scan fit model. We apply this framework to analyze the potential for microvilli structures, abundant in some cell lines, to perturb z-scan measurements and we outline data quality controls that contain the potential for error. This work provides a foundation supporting the use of simple stratified layers to model concentration profiles within the living cell together with checks that identify when such simplified models may be inapplicable for a given level of precision. This work has been supported by grants from the National Institutes of Health (R01 GM064589, R01 GM098550, RO1 GM124279).