The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell-matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization. mechanobiology | stem cell migration | stem cell differentiation | extracellular matrix | stiffness

Optical elastography, the use of optics to characterise and map the mechanical properties of biological tissue, involves measuring the deformation of tissue in response to a load. Such measurements may be used to form an image of a mechanical property, often elastic modulus, with the resulting mechanical contrast complementary to the more familiar optical contrast.Optical elastography is experiencing new impetus in response to developments in the closely related fields of cell mechanics and medical imaging, aided by advances in photonics technology, and through probing the micro-scale between that of cells and whole tissues. Two techniques have shown particular promise recently: optical coherence elastography and Brillouin microscopy; for medical applications, such as in ophthalmology and oncology, and as new techniques in cell mechanics.At every length scale, the mechanical properties of tissue are important. Mechanical and chemical interactions at the molecular and cellular level are fundamentally interwoven in determining biological function, and such interactions and related mechanical properties play an important role in the onset and progression of many diseases, including eye disease, cancer, and atherosclerosis 1,2 . Over the past 25 years, a range of elastography techniques have been developed to image the mechanical properties of tissue 3 . Elastography is now a commercial medical imaging technique, mainly finding application as a diagnostic tool in the assessment of liver fibrosis 4 , and of breast cancer 5 . Primarily based on ultrasonography or magnetic resonance imaging, such elastography provides images, known as elastograms, over centimetre to whole-body depth ranges, at millimetre-scale spatial resolutions far lower than is possible with optics. At higher resolutions, on the cellular scale, the measurement of mechanical properties underpins the field of cell mechanics 2 , which is focussed on understanding cellularscale mechanical properties and how cells respond to physical forces and the mechanical properties of their environment. Cell mechanics is supported by nano-and micro-imaging techniques, such as atomic force microscopy (AFM) and traction force microscopy 6 .Optical elastography is at a much earlier stage of development than the methods employed in medical imaging or in cell mechanics. It is ideally positioned to image mechanical properties on the intermediate scale, between that of cells and organs [7][8] , which presents new opportunities in the understanding, diagnosis and treatment of disease 6 . The use of optics in elastography offers the combination of micro-scale imaging, potential for in vivo deployment and high sensitivity to variations in mechanical properties, and holds promise for a wide range of applications in areas such as oncology, ophthalmology and cell mechanics. Many optical elastography techniques have been proposed, for example, based on optical coherence tomography 7-9 , Brillouin microscopy 10 , laser speckle 11 , ultrasound-modulated optical tomography 12 and dig...