1. 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-Golomb Tufts University SPIE Optics and Photonics 2013

2. 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 DuctAcini Dhimolea Biomaterials 2010

3. 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

4. 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 Gel Cell Media Glass Slide

5. 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

6. 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

7. 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 collagen gel at 28 mW F max x max σ

8. 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 k g or G’ 7.Must first calibrate for F max of trap at given power Control sample with known G’  assume homogeneity Measure displacement at each trap power  solve F max Restoring F gel x max σ

9. 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

10. 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/Matrigel Avg Diameter: 3.1 μm Collagen Avg Diameter: 3.7 μm

11. Test 3: Photobleaching Does Not Affect Image Size Ran 6 consecutive scans in collagen gel and observed bead image diameters  no real change

12. 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

13. 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 Slope w/Softness

14. 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 CollagenAcinus in Matrigel

15. 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

16. 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

17. 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

18. 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