This journal is © The Royal Society of Chemistry 2016 Integr. Biol. Cite this: DOI: 10.1039/c5ib00284b Deformability-based cell selection with downstream immunofluorescence analysis Josephine Shaw Bagnall,† a Sangwon Byun,†‡ a David T. Miyamoto, bc Joon Ho Kang, d Shyamala Maheswaran, be Shannon L. Stott, bfg Mehmet Toner eg and Scott R. Manalis* ahi Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell’s biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor’s record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell’s deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories. Insight, innovation, integration Although the mechanical properties of single cells have been associated with cell phenotype and malignancy, it has been difficult to directly correlate each cell’s biophysical characteristics to its molecular traits. Here, the integration of a cell sorting method with a suspended microchannel resonator enables precision measurements of single-cell buoyant mass and deformability to be correlated to each cell’s surface protein expression assessed by fluorescence imaging after off-chip collection. Using this technique, we sort and collect tumor cells from cell line samples, blood samples spiked with cell lines, and a metastatic prostate cancer patient blood sample based on their passage times through a constriction, providing insight regarding the relationship between each cell’s relative EpCAM expression to its distinct biophysical characteristics. Introduction Mechanical properties of single cells have drawn increasing attention in their ability to identify changes in cell phenotype, including those in differentiation and malignancy, and even to provide diagnostic value. 1–5 Various techniques used to probe the deformability of single cells include micropipette aspiration, 6,7 atomic force microscopy, 8,9 microrheology, 10–13 optical stretch- ing, 14,15 hydrodynamic deformation, 16,17 and microfluidic con- striction devices. 3,18–20 One such microfluidic device is the suspended microchannel resonator (SMR), which, based on the resonance frequency of the sensor, records the position of the center of mass of a cell with up to sub-micron precision as it passes through the constriction (Fig. 1A and B). The resonance frequency signal enables the measurement of the passage time of a cell through the constriction, and can be used to parse out a Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. E-mail: srm@mit.edu b Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA c Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA d Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA e Department of Surgery, Massachusetts General Hospital, Harvard Medical School, BostonMA, USA f Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA g Massachusetts General Hospital Center for Engineering and Medicine, Harvard Medical School, Boston, MA, USA h Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA i Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA † Equal contribution. ‡ Current address: Bio-Medical IT Convergence Research Department, Electronics and Telecommunications Research Institute, Daejeon, Republic of Korea. Received 9th November 2015, Accepted 5th March 2016 DOI: 10.1039/c5ib00284b www.rsc.org/ibiology Integrative Biology TECHNICAL INNOVATION Published on 11 March 2016. Downloaded on 21/03/2016 15:19:55. View Article Online View Journal