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Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Spying on Cells: Cellular and Spying on Cells: Cellular and Subcellular Subcellular Analysis using Novel Polymeric Micro Analysis using Novel Polymeric Micro-

• and

and Nanostructures Nanostructures Xin Zhang Xin Zhang Associate Professor Associate Professor

Boston University Boston University US US-

• Korea

Korea Nano Nano Forum Forum April 2008 April 2008

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Road Map of Nanobio Road Map of Nanobio-

• sensors

sensors

Cellular force Ion channel activity Gene expression Nanoelectrical sensors Cell adhesion Nano-optical sensors Nanomechanical sensors

• 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.

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Polymer Pillar Array Polymer Pillar Array

Low Aspect Ratio High Aspect Ratio

Spacebars indicate 5 µm

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Realization of 3D Realization of 3D Structures Structures

• Utilizing the micromolding

process, complex structures, varying in both lateral dimension and height, are fabricated.

• Elevated sidewalls are to

provide vertical surfaces for cell attachment.

• This may avoid the artificial

polarization of cells induced by conventional dishes, thus allowing a more in-vivo-like cellular morphology.

• Polymeric posts placed

between the sidewalls are to further enhance cell attachment.

Embedded Pillars Sidewalls Posts 10 µm 10 µm

Replicated from the same master template

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Experimental Setup for Cellular Force Experimental Setup for Cellular Force Measurement Measurement

Vacuum pump Inverted microscope CCD camera Computer system for imaging analysis Buffer solution Liquid pump Waste solution Feedback control Thermometer Heating rod Perfusion chamber Inlet Outlet

x y z

O

Electrical contact pair

x y z

O

+

• Outlet

Inlet Thermometer probe PDMS chip

37°C; Real time; Live cell; CO2 preferable gas concentration

Fluidic Connection Electrical Connection Inverted Microscope

• The cardiac myocytes

were isolated from Wistar rats

• The cells were plated
• n the fabricated

structures

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Myocyte PDMS pillars

10 µm

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

The underlying pillar has periodical displacement with the cell contraction The pillar away from the cell does not represent a

• bvious periodicity. The

displacement is on the noise level.

F F

46 48 50 52 54

Myocyte Pillars

44 Time (s) Length (µm) 10 12 14 16 18 20 32.8 33.0 33.2 33.4 31.94 31.98 32.02 32.06 62.0 58.0 66.0 230 222 224 226 228 B C A B C

10 µm

Conditions

• The isolated myocytes were plated
• n a PDMS substrate with pillars of

aspect ratio 2:1, allowing 24 hours for adhesion.

• The myocytes were stimulated by a

digital pacer with a periodical voltage pulse (DC 20V at 0.5 Hz), which provided an additional electrical potential besides the action potential

• f the myocytes to activate the

contractile proteins.

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Image Processing for Cellular Image Processing for Cellular Force Measurement Force Measurement

Extract and remove background nonuniformity Applying thresholding to the image Locate individual pillars Compare derived pillars array with a reference Derive the deformation map and force map

150 nN 5 µm

(a) (b) (c) (d)

1.00 2.00 3.00 4.00

5 10 15 20 25 30 35 40 45 50

Area of pillar top (µm2) Pillar number

Residual noise from the cell Residual noise from the cell

Image Processing Binary array Histogram

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Contraction Force Analysis Contraction Force Analysis

Cellular force (nN)

1 2 130 140 150 160 170 180

Time (s) (c)

2 1

Time (s) (d)

20 30 50 60 70 40

x (µm)

200 400 600 800 1000 1200

• 200
• 160
• 120
• 80
• 40

40 80 120 160 200 200 300

y (µm) Cellular force (nN) (x component) (a)

100 200 200 400 600 1000 1200

x (µm)

100 200 300

y (µm) Cellular force (nN) (y component) (b)

800

• 200
• 160
• 120
• 80
• 40

40 80 120 160

Force component along contraction axis Force component along transverse axis

Force measurement with subcellular resolution Force evolution measured in real time

The force evolution reveals the alignment of motile units in cardiac myocyte, which conforms to the physiologic fact.

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Chemical Sensing

– Drug evaluation – Cell mechanics study – Pathology investigation – etc.

Force Evolution during Force Evolution during Chemical Perfusion Chemical Perfusion

Validation of the inotropic effect of the cardiac myocytes in response to the β-adrenergic stimulation Currently validated by an increase of inward calcium current, a greater rate of release of calcium ions from the sarcoplasmic reticulum (SR), and an accelerated reuptake of calcium into the SR

Time (s) 299.2 299.4 299.6 299.8 300 300.2 300.4 300.6 300.8 15.0 15.1 15.2 40.2 40.4 40.6 40.8 41.0 41.2 41.4 41.6 41.8 15.0 15.1 15.2 (b) (a) ~ 21.2 nN ~ 29.8 nN Displacement (µm)

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

F δ

Nanoscale Nanoscale Biomechanosensor Biomechanosensor

• 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

? How to measure the deformation in nanostructures?

• Direct optical measurement is no longer appropriate

) sin( n 2 / α λ

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

5 µm

SEM of fabricated equally spaced polymeric SEM of fabricated equally spaced polymeric periodic substrate (PPS) periodic substrate (PPS)

5µm

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Imaging Imaging Interface: Interface: Optical Optical Moir Moiré é technique technique

* 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 Force Mapping Mapping in in Vascular Vascular Smooth Smooth Muscle Muscle Cells Cells

0 hr 4 hrs 8 hrs 12 hrs 18 hrs 24 hrs 0 hr 4 hrs 8 hrs 12 hrs 18 hrs 24 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

• n the substrate.

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Corresponding Corresponding Force Force Map Map Derived Derived from from Moir Moiré é

2 4 6 8 2 4

B C

Derived cellular traction force mapping * Length and direction of the arrows: the direction and magnitude of the forces derived from the map * Colors: the magnitude of the displacements * Decrease of traction force with decreasing spreading area * Concentrated at the boarder of the cells, pointing to the nuclei directions * Least traction forces concentrated at the central region of the cells

12 hrs 18 hrs 24 hrs

2 4 6 8 2 4

A

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Determining Force Evolutions from Moir Determining Force Evolutions from Moiré é Patterns Patterns

1 2 3 4 5 5 µm

4 8 12 16 20 24

• 10

10 20 30 40 50

Culture time (hr)

Force (nN) Point 1 Point 2 Point 3 Point 4 Point 5

To determine the magnitude of the contraction force developed on adhesion areas from the moiré patterns: * Derived the traction force on five locations * Mapped the evolution of the traction forces over time

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Conclusions Conclusions

Fabrication

– Polymer micro- and nanostructures with various aspect ratios Characterization – Deep beam model – Moiré techniques Measurement and Analysis – Micro/nanofabricated polymer based system provides in-vitro cell traction force mapping in the sub-cellullar level – Optical moiré approach provides robust and real-time imaging of in-plane cell traction force mapping

• Such optical moiré system can be readily employed to study migration,

morphology, motility and many other cell-substrate mechanical interactions

• n patterned polymer substrates.
• Since our approach requires neither the tracking/monitoring nor the

visibility of each individual pillar, we anticipate that this method will increasingly find more applications in micro and nano patterned substrates for a variety of mechanics studies in living cells.

Laboratory for Microsystems Technology, Boston University Laboratory for Microsystems Technology, Boston University

Acknowledgements Acknowledgements

• NSF CAREER Award
• Brigham and Women's Hospital,
• Tufts-New England Medical Center
• Vanderbilt Medical Center