Evaluating cell matrix stiffness with a multiphoton confocal microscope-optical tweezer setup

Berney Peng, Carlo Alonzo, Lawrence Xia, Lucia Speroni, Irene Georgakoudi, Ana Soto, Carlos Sonnenschein

and Mark Cronin-GolombTufts University

SPIE Optics and Photonics 2013

Introduction• Cell matrix stiffness is central in physiological processes

such as breast epithelial morphogensis– Extracellular matrix (ECM) stiffness epithelial structure

formation collagen fiber organization

• Breast carcinogenesis potentially affected by tissue stiffness– Mammographic density is a key risk factor– local fiber organization on local viscoelasticity important

Therefore, it is necessary to assess ECM organization through imaging in the context of stiffness

Duct Acini

Dhimolea Biomaterials 2010

Highlights• Description of multiphoton-optical tweezer setup

– 2D Linear scanning method of tweezer to measure stiffness

– SHG, TPEF, confocal reflectance imaging channels

• Primary results:– Proof of concept, repeatability, and control tests

• Resolve stiffness in different materials• Trapping power vs. deformation

– Stiffness around acinar and ductal epithelial structures

Speroni Tissue Eng C 2013

MCF10A in Collagen

SHG of T47D cells

Study System: Collagen and Matrigel Cell Cultures

• Type of Cell: MCF10A normal breast epithelial cell• Two types of 3D cultures:

– 1 mg/ml Collagen Type I, rat tail• Promotes ductal structures

– 1 mg/ml Collagen Type I, rat tail + 50% Matrigel• Promotes acinar structures

– Gels embedded with 2 μm diameter fluorescent beads at 0.01% w/v

3D attached gel culture

Collagen GelCell Media

Glass Slide

Multiphoton-Optical Tweezer Setup

Imaging:• 800 nm, 100fs

Ti:Sapphire Laser• 40x objective, 1.1 NA• 400x400 μm imaging

region• 400, 460, 525 nm PMT

channels + reflectance

Trapping:• x-y scanning galvos• Bead TPEF signal

detected by Labview• Scan steps ~0.2 μm,

scan velocity ~9-10 μm/s

Process of Imaging and Stiffness Measurement

• High speed imaging beam on raster scan• Objective mounted to stepper motor z-axis• Obtain z-stack image• Find bead of interest and perform 2D linear scan

Acinus, Volume View

Process of Microscale Stiffness Measurement

1. 2D linear scan over bead at fixed z-plane

2. Tweezer creates TPEF and pulls at bead• Force profile of beam

• Maximum beam force

3. Deformation More tweezer-bead overlap

TPEF bead image:4 z-slices of collagengel at 28 mW

Fmax

xmax

σ

Process of Microscale Stiffness Measurement

4. Larger TPEF image

5. Measure displacement relative to fixed bead

6. Calculate elastic modulus:• Given a gel balancing force, Hookean assumption:

• Determine kg or G’

7. Must first calibrate for Fmax of trap at given power• Control sample with known G’ assume homogeneity

• Measure displacement at each trap power solve Fmax

Restoring Fgel

xmax

σ

Test 1: Gelatin Deforms More at Higher Trap Power

• 2D Linear scan over same bead in same material at same power more deformation at larger power

• Used 0.07g/ml gelatin gel• Scan at 40 mW and 84 mW, 0.2 μm/step resolution

40 mW 84 mW

Gelatin

Horizontal—2.9 μm

Vertical—3.6 μm

Horizontal—3.3 μm

Vertical—3.9 μm

Test 2: Collagen Softer than Matrigel Culture

• 2D Linear scan over bead in different material at same power more deformation in softer material

• Used 1 mg/ml Collagen gel and Matrigel gel• Scan at 65 mW, 0.2 μm/step resolution

• Obtained expected result– Lack of spherical 2D image shape due to local anisotropy?

Collagen/MatrigelAvg Diameter: 3.1 μm

CollagenAvg Diameter: 3.7 μm

Test 3: Photobleaching Does Not Affect Image Size

• Ran 6 consecutive scans in collagen gel and observed bead image diameters no real change

Test 4: For Stationary Bead, Image Size Unchanged

• A fixed bead should possess identical bead image diameters for all laser powers size is power invariant

• Scans were performed on a 3.5 mg/ml collagen gel, no cells– Powers of 10, 20, and 40 mW, 5 samples/power

Test 5: Linear Relation Between Power and Image Size • Measurements on 1mg/ml collagen gel at various

powers– 10, 20, 40 , 60, and 80 mW– At least 5 samples per laser power

Increasing Slopew/Softness

Experiment: Stiffness Near and Away from Structures• Cultured breast epithelial cells, MCF10A in two types of

cultures– 1mg/ml collagen gel only

• Observed more ductal structures

– 1mg/ml collagen gel with 50% Matrigel by volμme• Observed more acinar structures

– Laser power, 80 mW– 3 weeks post seeding– Near and far from structure, both ductal and acinar

Duct in Collagen Acinus in Matrigel

Cell Matrix Softer Near Ductal Cells Than Far

• Cell matrix is statistically softer near the duct than away• No significant stiffness difference (near vs. far) in acinar

* NS

Cell Matrix More Variable Near Ductal Cells Than Far• Cell matrix is statistically more variable near the duct

than away• No significant stiffness difference (near vs. far) in acinar

more homogeneous

Ductal structures seem to remodel collagen differently than acinar structures

*NS

Conclusions• Viability of 2D linear scanning method

– Detected differences between two different materials– Positive linear trend in trapping power and deformation– Interesting biological questions raised from Acinar vs. Ductal

stiffness study• Compare near and away from cells in same culture

• Future Work– Calibrate Max Force vs. laser trapping power– Quantify collagen fiber organization (Speroni Tissue Eng C

2013) and correlate to stiffness– Examine 2D and 3D isotropy– Develop oscillatory technique

Acknowledgements• Personnel

– Greg Whitt

• Funding– Avon Grant 2-2009-093 and 02-2011-095 to AMS– NIEHS/NIH ES 08314 to AMS– American Cancer Society Research Scholar Grant RSG-09-

174-01-CCE to IG