articleLive cell flattening — traditional and novel approaches

Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field (BF) to total internal reflection fluorescence (TIRF) microscopy. Fundamental processes, such as mitosis and in vivo actin polymerization, have been investigated using these techniques. Here, we review the well known agar overlayer protocol and the oil overlay method. In addition, we present more elaborate microfluidics-based techniques that provide us with a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method. PACS Codes: 87.64.-t, 47.61.-k, 87.80.Ek 1. Background Studies of biochemical reactions are traditionally carried out on large cell populations, typically on the order of a million cells or more. Based on the biochemical data from such studies, systems biology has greatly advanced our knowledge of metabolic pathways and signaling cascades in living cells [1]. Yet, as our understanding of biochemical interaction networks becomes more complete, it is advantageous to complement this progress with fluorescence microscopy experiments that characterize the dynamics of such pathways on the single cell level. With fluorescence microscopy, we can observe the expression levels and localization of tagged proteins in each cell. One also can examine the properties of and interactions between proteins using Fluorescence Correlation Spectroscopy and Förster Resonance Energy Transfer. Some of the limitations in live cell microscopy are due to the movement of cells in and out of the plane of focus. To overcome these limitations, cells can be flattened to quasi twodimensional geometries, ensuring that the cells remain in the focal plane. These flattened geometries have been advantageous for observing the eukaryotic cytoskeleton during chemotaxis and cytokinesis [2,3]. Recent examples combined cell flattening with Total Internal Reflection Fluorescence (TIRF) microscopy to increase the area of observation and keep the cell membrane and cortex in the evanescent field [4]. In this article, we present a brief review of established overlay approaches for cell flattening. Furthermore, we introduce microfluidic techniques to achieve greater control over the degree of flattening. Each technique is exemplified with a short demonstration using cells of the social amoeba Dictyostelium discoideum [5]. We discuss the advantages and disadvan* Correspondence: beta@uni-