Spying on Cells: Cellular and Subcellular Subcellular Spying on Cells: Cellular and Analysis using Novel Polymeric Micro- - and and Analysis using Novel Polymeric Micro Nanostructures Nanostructures Xin Zhang Xin Zhang Associate Professor Associate Professor Boston University Boston University US- -Korea Korea Nano Nano Forum Forum US April 2008 April 2008 Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Road Map of Nanobio- -sensors sensors Road Map of Nanobio • How can we best monitor living cells in-situ and continuously to understand, characterize, and model functional behavior at the cellular levels so as to explore biosensor specificity and flexibility for distinct responses to different combinations of stimuli? • Many key problems in biochemical sensing can be solved by converting biological or chemical response to an electrical, optical, or mechanical signal using micro/nanosystems. • The use of living cells as sensor elements provides the opportunity for high sensitivity in a broad range of biologically active substances and physical stimuli that affect cell responses. Nano-optical sensors Nanoelectrical sensors Gene expression Ion channel activity Cellular force Nanomechanical sensors Cell adhesion Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Polymer Pillar Array Polymer Pillar Array High Aspect Ratio Low Aspect Ratio Spacebars indicate 5 µ m Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Realization of 3D Realization of 3D Structures Structures • Utilizing the micromolding Replicated from process, complex the same master structures, varying in both template lateral dimension and height, are fabricated. • Elevated sidewalls are to provide vertical surfaces for cell attachment. This may avoid the artificial o polarization of cells 10 µ m induced by conventional dishes, thus allowing a more in-vivo-like cellular 10 µ m Embedded Pillars Sidewalls morphology. • Polymeric posts placed between the sidewalls are to further enhance cell attachment. Posts Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Experimental Setup for Cellular Force Experimental Setup for Cellular Force Measurement Measurement Feedback control • The cardiac myocytes were isolated from Liquid pump z Wistar rats y x • The cells were plated Heating Thermometer O rod Electrical on the fabricated Vacuum contact pair Inlet pump structures Outlet � Fluidic Connection Perfusion chamber Buffer solution � Electrical Connection z � Inverted Microscope + - y x Waste solution O Inverted microscope Inlet Outlet CCD camera Computer system Thermometer for imaging analysis PDMS chip probe 37 ° C; Real time; Live cell; CO 2 preferable gas concentration Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
PDMS pillars Myocyte 10 µ m Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Deformation Isolation between Cells Deformation Isolation between Cells and the Base Substrate and the Base Substrate Myocyte Pillars Conditions - The isolated myocytes were plated on a PDMS substrate with pillars of aspect ratio 2:1, allowing 24 hours for adhesion. B - The myocytes were stimulated by a 10 µ m digital pacer with a periodical voltage C pulse (DC 20V at 0.5 Hz), which provided an additional electrical 66.0 potential besides the action potential A 62.0 of the myocytes to activate the Length ( µ m) contractile proteins. 58.0 222 224 226 228 230 The underlying pillar has 33.4 periodical displacement B 33.2 with the cell contraction 33.0 F 32.8 10 12 14 16 18 20 The pillar away from the 32.06 cell does not represent a 32.02 C F obvious periodicity. The 31.98 displacement is on the 31.94 noise level. 44 46 48 50 52 54 Time (s) Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Image Processing for Cellular Image Processing for Cellular Force Measurement Force Measurement (a) (b) 5 µ m 150 nN (c) (d) Image Processing Binary array Histogram � Extract and remove background 50 nonuniformity 45 � Applying thresholding to the 40 35 image Pillar number 30 � Locate individual pillars 25 20 Residual noise Residual noise � Compare derived pillars array 15 from the cell from the cell with a reference 10 5 � Derive the deformation map and 1.00 2.00 3.00 4.00 Area of pillar top ( µ m 2 ) force map Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Contraction Force Analysis Contraction Force Analysis Force evolution measured in Force measurement with subcellular resolution real time Force component along Force component along contraction axis transverse axis 200 200 160 Cellular force (nN) Cellular force (nN) 160 120 (x component) 120 (y component) 80 80 40 40 0 0 -40 -40 -80 -80 -120 -120 -160 -160 -200 -200 300 300 200 200 y ( µ m) The force evolution reveals the y ( µ m) 100 100 800 1000 1200 1200 alignment of motile units in cardiac 600 1000 (a) 400 800 200 400 600 0 200 0 (b) x ( µ m) x ( µ m) myocyte, which conforms to the physiologic fact. Cellular force (nN) 180 70 170 60 160 50 150 40 140 30 130 20 1 2 1 2 (c) (d) Time (s) Time (s) Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Force Evolution during Force Evolution during Chemical Perfusion Chemical Perfusion � Chemical Sensing � Validation of the inotropic effect of the cardiac myocytes in response to the β -adrenergic – Drug evaluation stimulation – Cell mechanics study � Currently validated by an increase of inward – Pathology investigation calcium current, a greater rate of release of calcium ions from the sarcoplasmic reticulum – etc. (SR), and an accelerated reuptake of calcium into the SR 15.2 15.1 ~ 21.2 nN Displacement ( µ m) 15.0 40.2 40.4 40.6 40.8 41.0 41.2 41.4 41.6 41.8 (a) 15.2 15.1 ~ 29.8 nN 15.0 (b) 299.2 299.4 299.6 299.8 300 300.2 300.4 300.6 300.8 Time (s) Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Nanoscale Biomechanosensor Biomechanosensor Nanoscale δ F • It is sensible to downsize the microfabricated structures to nanoscale: – To enhance the probing sensitivity – To enhance the spatial resolution – To improve the material compatibility • Direct optical measurement is no longer appropriate λ α / 2 n sin( ) ? How to measure the deformation in nanostructures? Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
SEM of fabricated equally spaced polymeric SEM of fabricated equally spaced polymeric periodic substrate (PPS) periodic substrate (PPS) 5 µ m 5 µ m Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
é technique Moir é Imaging Interface: Interface: Optical Optical Moir technique Imaging * Moiré Fringes: or the moiré effect refers to light/dark bands seen by superimposing two nearly identical arrays of lines and dots. * In most basic form, moiré methods are used to measure displacement fields. Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Force Mapping Mapping in in Vascular Vascular Smooth Smooth Muscle Muscle Cells Cells Force 0 hr 0 hr 4 hrs 4 hrs 8 hrs 8 hrs 24 hrs 24 hrs 18 hrs 18 hrs 12 hrs 12 hrs As the VSMCs spread out in DMEM media with serum , the moiré patterns changed from regularly distributed to locally distorted, and further resembled a natural centrifugal pattern, revealing the concentric profile of the traction forces developed on the substrate. Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
Corresponding Force Force Map Map Derived Derived from from Moir Moiré é Corresponding 4 Derived cellular traction force mapping 2 * Length and direction of the arrows: the direction and 0 magnitude of the forces derived from the map * Colors: the magnitude of the displacements 8 4 6 2 4 0 A 2 12 hrs 8 6 4 B 2 18 hrs * Decrease of traction force with decreasing spreading area * Concentrated at the boarder of the cells, pointing to the nuclei directions C * Least traction forces concentrated at the central region of the cells 24 hrs Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University
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