1 2 Wedged AFM-cantilevers for parallel plate cell mechanics 3 Martin P. Stewart 1 Q1 , Adrian Hodel 1 , Andreas Spielhofer, Cedric J. Cattin, Daniel J. Muller, Jonne Helenius ⇑,1 4 D-BSSE, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland 5 6 8 article info 9 Article history: 10 Available online xxxx 11 Keywords: 12 AFM 13 Cantilever 14 Cantilever functionalization 15 Cantilever modification 16 Wedge 17 Wedging 18 Cell mechanics 19 Parallel plate 20 Cell compression 21 Mitotic cell mechanics 22 Cell rounding Q2 23 24 abstract 25 The combination of atomic force microscopy (AFM) and optical microscopy has gained popularity for 26 mechanical analysis of living cells. In particular, recent AFM-based assays featuring tipless cantilevers 27 and whole-cell deformation have yielded insights into cellular function, structure, and dynamics. How- 28 ever, in these assays the standard 10° tilt of the cantilever prevents uniaxial loading, which complicates 29 assessment of cellular geometry and can cause cell sliding or loss of loosely adherent cells. Here, we 30 describe an approach to modify tipless cantilevers with wedges and, thereby, achieve proper parallel 31 plate mechanics. We provide guidance on material selection, the wedge production process, property 32 and geometry assessment, and the calibration of wedged cantilevers. Furthermore, we demonstrate their 33 ability to simplify the assessment of cell shape, prevent lateral displacement of round cells during com- 34 pression, and improve the assessment of cell mechanical properties. 35 Ó 2013 Published by Elsevier Inc. 36 37 38 39 1. Introduction 40 The physical properties of cells are fundamental to their 41 function. One technique to investigate the mechanical properties 42 of isolated cells and tissue aggregates is the use of parallel micro- 43 plates. In the case of low-adherence samples this approach allows 44 the internal pressure and surface tension to be determined [1]. Fur- 45 thermore, adherent samples can be both stretched and compressed 46 to study the relationship between load and deformation [2]. Indeed, 47 several of the first examinations of single cell mechanics were 48 conducted with parallel plate compression setups on egg cells from 49 marine organisms [1,3–6]. Since then parallel plate assays have 50 been further developed and implemented to gain insight into the 51 mechanics, structure and function of tissue aggregates [7–9], and 52 both isolated plant [10], and animal cells [2,11–20]. The setups vary 53 from custom-built micromanipulator type devices [11,18,20] to 54 commercially available atomic force microscopy (AFM), where a 55 tipless cantilever acts as the upper microplate for performing cell 56 deformations and measuring forces [2,17,21]. One issue that has 57 prevented the wider use of AFM is the standard 8–12° tilt of the 58 cantilever, which obfuscates uniaxial loading. In this paper we will 59 outline a simple method to correct this problem by molding an 60 adhesive wedge onto tipless cantilevers. We expect this basic 61 advancement to pave the way for the wider use of AFM in parallel 62 plate mechanics experiments. 63 AFM, originally developed to image surfaces [22], is used to 64 probe the mechanical properties of a wide range of native biolog- 65 ical samples in physiologically relevant environments [23]. These 66 range from tissues, cells, membranes, and protein assemblies, to 67 single proteins and nucleic acids [24]. The heart of any AFM system 68 is a spring-like flexible cantilever, the movement of which is 69 monitored by a laser-based optical lever setup [25], which enables 70 the detection of Ångström-scale cantilever deflections and forces 71 ranging from picoNewtons to microNewtons. In the context of cell 72 mechanics, the choice of cantilever geometry is a critical parameter 73 in the experimental design. For example, cantilevers with sharp 74 tips can be used to probe nanometer scale regions of the cell 75 surface while flat tipless cantilevers are better suited to probe 76 whole-cell mechanics [21]. To provide sufficient clearance, 77 standard AFM systems mount the cantilever at an angle of 8–12° 78 relative to the sample surface. This angle prevents proper parallel 79 plate mechanics and uniaxial loading (Fig. 1A Q3 ). One problem 80 caused by this is lateral instability (sliding) of weakly adherent 81 cells, especially when subject to larger compressions. A second 82 problem is the asymmetric cell shape, which complicates the 83 determination of important geometrical parameters such as 84 cell-cantilever contact area, cell surface area, and cell volume. In 85 order to solve these problems and perform proper AFM based 86 parallel plate mechanics, we developed a method of modifying 1046-2023/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ymeth.2013.02.015 Abbreviations: AFM, atomic force microscopy; SEM, scanning electron micros- copy; UV, ultraviolet; PDMS, polydimethylsilane; PTFE, polytetrafluoroethylene; DMEM, Dulbecco’s modified Eagle medium; DIC, differential interference contrast. ⇑ Corresponding author. Fax: +41 61 387 3982. E-mail address: jonne.helenius@bsse.ethz.ch (J. Helenius). 1 These authors contributed equally to this work. Methods xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth YMETH 3064 No. of Pages 9, Model 5G 11 March 2013 Please cite this article in press as: M.P. Stewart et al., Methods (2013), http://dx.doi.org/10.1016/j.ymeth.2013.02.015