Tissue Loading Micro-Chamber

1. Tissue Loading Micro-Chamber Flow-Through Environment for High-Power Microscopic Observation Aaron Desjarlais Jessica Kornfeld Michael Lee Matthew McGrath Jeff Perry

2. Problem Statement • Mechanism to apply small uniaxial load to live tissue sample • Record strain, load, displacement while operation is underway • Provide interface with the Nikon TE2000E Inverted Optical Microscope • Temperature-controlled environment • Provide for media flow through device to sustain specimen for long time periods

3. Background - Collagen • Collagen is the most abundant protein on Earth • Structural molecule of choice for vertebrates • Bears and transmits tensile loads applied to the body • Connective collagenous tissue naturally degenerates • Decline of usable collagen-based tissue • Increase in growing tissue in the laboratory environment

4. Background - Project • Collagenous tissue adapts to its environment • When applying a load, extracellular matrix remodels in response to strain • Difficult to observe in vivo due to need for high-power objectives

5. Literature Search • Instron Planar-Biaxial Soft Tissue Test System Instron - BioPuls Submersible Pneumatic Grips and Temperature-Controlled Bath Bose BioDynamic test systems Capstone Project (Spring 2005) Refined by Kelli Church for master’s thesis (Spring 2007) Bioptechs Chamber

6. Requirements - Specifications • Must be attachable to Nikon TE2000E stage • Placement: ≤200μm from objective lens • Micro-chamber volume: <200 micro-liters • Temperature: 37°C ± 0.5° • Tissue size: 1cm x 1cm x ~10 - 1000μm • Uni-axial Load: minimum ~0.1N • Strain Accuracy: ± 1μm • Record displacement, load, strain, and temperature

7. Prototype Mounted on Microscope

8. Proposed Design

10. Linear Guide System

11. Guide System - Design Constraints • Linearity • The system must remain in camber • Rigidity • The system must be rigid enough so any displacement in the system does not add error to the measurement • Size constraint • Must fit under the condenser of the microscope

12. Micro-Chamber Base

13. Micro-Chamber Base - Design Constraints • Rigidity • The system must be rigid enough so any displacement in the system does not add error to the measurement • Size • The base must interface with existing mounting location on the microscope stage

14. Micro-Chamber - Interior

15. Material Selection 316 Stainless Steel • High thermal conductivity:  • 16.3 W/m-K (113 BTU-in/hr-ft²-°F) • Operate at environmental temperature • Melting Point: 1370 - 1400 °C (2500 - 2550 °F) • Corrosion resistant • Can survive repeated common sterilization methods

16. Micro-Chamber Exterior

17. Material Selection Polycarbonate • Low thermal conductivity • 0.142 - 0.26 W/m-K (0.985 – 1.8 BTU-in/hr-ft²°F) • Corrosion resistant • Sterilization • Compatible with common clinical disinfectants; isopropyl alcohol (rubbing alcohol) • Low permeability • Water absorption: 0.0500 - 0.700%

18. Micro-Chamber Bacteria Sealing • Dynamic elements: two seals with antibacterial solution injected in between both seals • Static elements: single seal or gasket through compression to fill the gap • 0.1 pounds of force is required per bolt on the Top Cover

19. Drive/Sensor System

20. Drive System Design Concepts Pulley Drive Mechanism Rack and Pinion Mechanism Direct Drive Mechanism Direct Drive with Spring Mechanism

21. Linear Actuator Chosen Motor: Zaber Technologies T-LA60A • Accuracy: 0.1 µm • Already used on existing system • Has manual control to ease setup • Holds up to 15N continuous load Requirements: • Control and measure strain to 1 µm • Allow for minimum 10mm of travel

22. Requirements: Uni-axial Load: minimum ~0.1N Miniature Submersible Corrosion resistant Selected Load Cell: Sensotec Model 31 Load capacity range from 50 g to 500 g (0.5 N to 5 N) 17-4 PH stainless steel Force Transducer

23. Control System • LabView Control System • Capable of Load or Strain Control • Adjustable PID Parameters

24. Control System NI Labview Data Acquisition • PID Toolkit • Uses existing interface as baseline • Signal conditioning card

25. Load Control Tests • Used Med-4720 silicone elastomer as specimen • Tested using 0.1N as required load • Max. error of ± 0.002N once steady state reached

26. Tissue Grip Mechanism

27. Grip Design • Requirements: • Apply even clamping pressure • Easy to operate • Accommodate specimens up to 10mm wide, 10 μm to 1000 μm thick • Must be small to minimize chamber volume • No part of the clamp must be lower than the sample

28. Grip Design Hinged Clamp Sliding Plates Sliding Bar Clamp

29. Grip Design Chosen Design: Sliding Bar Clamp

30. Thermal Control

31. Selected Temperature Controller • Omega CNi 3222-C24 • AutoTune PID High Accuracy ±0.5°C (0.9°F), 0.03% Reading • 2 Outputs • Dual Alarm • Universal Input - Accepts all t/c and RTD’s • PC RS-232 output • Free Software • Ramp to Setpoint • Cartridge Heaters • Do not restrict design evolution • Versatile, easily mounted • Compact Design • Powerful Element From Spring 2005 Capstone Design Group

32. Previous System Cartridge heater Embedded in Copper Block Quartz glass environmental Chamber Drawings From Spring 2005 Capstone Design Group

33. Grips Stainless Inner Lining Insulating Plexiglas Copper Blocks Cartridge Heater Fluid Flow Silicone Tubing Entering Fluid Entering Fluid Exiting Fluid Heating System Components

34. Heat Transfer Model Comparison Previous Model Present Model Rcond,conv,fin (Top) Rcond,conv (Top) Rcond,conv, fin (Back) Rcond,conv,fin (Front) Rcond,conv (Back) Rcond,conv (Front) UP UP Rcond,conv – Robj cond, conv (Bottom) Rcond,conv,fin (Bottom)

35. Temperature Controller Testing

36. Temperature Testing Results

37. Future Work • Finish machining parts to specifications, primarily the adjustable piston • Program system to operate in parameters of strain control • Fully functional testing including, cornea loading and fluid heating assessment • Micro-Chamber volume minimization

38. Questions?

39. Backup Slides

40. Linear ActuatorTrade Study From Spring 2005 Capstone Design Group

41. Heat Source From Spring 2005 Capstone Design Group

42. Temperature ControllerTrade Study From Spring 2005 Capstone Design Group

43. Results of Heat Transfer Analysis: Heating Chamber From Spring 2005 Capstone Design Group

44. 1∙ 2∙ 3∙ ∙4 ∙5 *35 *34 33* 11∙ 12∙ *32 13∙ ∙15 ∙14 21∙ 22∙ *31 23∙ ∙25 ∙24 Nodal Analysis • System Divided into 37 Nodes, 6 fluid Nodes • Boundary Conditions: • System begins at ambient temperature in air • Fluid enters at room temperature and exits into large reservoir at room temperature • System is symmetrical on either side of the chamber. From Spring 2005 Capstone Design Group

45. Equivalent Circuit Analysis 1/hA (kA/L)ins (kA/L)cu T∞, h∞ (Top face) (Left face) (Back face) Equivalent Circuit for Node 1 qin= qout + qstored (ρcV)cu∙dT = ΣCij∙(Tji) + (q” ∙A) dt (ρcV)cu∙ΔT = ΣCij∙(Tji) + (q” ∙A) Δt ΔT = Δt ∙[ΣCij∙(Tji) + (q” ∙A)] (ρcV)cu Tf -Ti = Δt ∙[ΣCij∙(Tji) + (q” ∙A)] (ρcV)cu Tf = Ti + Δt∙[ ΣCij∙(Tji) + (q” ∙A)] Equivalent Circuit for Node 31 Ein = Eout +Estored qin = qout +qstored , q= qcond + qe (qc + qe)in= (qc + qe)out+ qstored (ρc)l ∙ ve ∙ Af ∙ dT/dt = q31-32+ q31-cu+ qe (ρc)l ∙ ve ∙ Af ∙ ΔT/Δt = q31-32 + q31-cu + qe ΔT = Δt / (ρc)l ∙ ve ∙ Af ∙ (q31-32 + q31-cu + qe) Tf-Ti = Δt / (ρc)l ∙ ve ∙ Af ∙ (q31-32 + q31-cu + qe) Tf = T31 + Δt / (ρc)l ∙ ve ∙ Af ∙ (q31-32 + q31-cu + qe) Tf = T31 + Δt / (ρc)l ∙ ve ∙ Af ∙ (C31-32 ∙(T32-T31)+ q31-cu (ρc)l ∙ ve ∙ Af /2∙(T32-T∞)) q”heater C12=kA/L 1 2 (right face) C16=kA/L 6 (front face) C1-11 11 (bottom face) 32 ρcveAf(T32+T31)/2 1/hlAf 2πrcukcu/ln(rcu/ro) 22 2πrcukcu/ln(rcu/ro) 23 27 28 ρcveAf(T∞+Ti)/2 ri ro rcu T∞, h∞ From Spring 2005 Capstone Design Group

46. Results of Heat Transfer Analysis: Chamber Fluid From Spring 2005 Capstone Design Group

47. Results of Heat Transfer Analysis: Tubing Fluid Temperature From Spring 2005 Capstone Design Group

48. Translation Block Stress Analysis

49. Base Stress Analysis

50. Base Deflection Analysis