1. Live Cell Imaging Chamber Andrew Dias, Justin Lundell, Val Maharaj, Jeremy Schaefer, Mike Socie Client: Lance Rodenkirch, UW School of Medicine Advisor: Prof. Bill Murphy

2. Outline Problem Statement Background Current Technology Design Constraints Design Alternatives Future Work

3. Problem Statement Design an imaging chamber that: – Controls gas composition in cell environment – Maintains constant temperature – Is compatible with Nikon Eclipse TE 2000 microscope – Has perfusion capabilities

4. Background - Need Examining some proteins is not possible in dead cells Medium for live cells requires controlled environment to maintain correct pH Growing cells require controlled temperature and delivery of nutrients (perfusion)

5. Background – Current Technology Incubator 2000 – 20/20 Technology Inc. – Allows temp. control from -10 to 60°C – Has atmosphere control option Focht Chamber System 3 – Bioptechs Inc. – Atmosphere and temp. control – Perfusion options – Gas flow speed control

6. Design Constraints Chamber must: – Maintain 95% air, 5% CO 2 atmosphere composition – Maintain temperature of 37.1°C – Fit on 30 cm x 27.6 cm microscope stage – Allow perfusion tubes to access cells

7. Design 1: Mixed Air Tank Imaging Chamber Hot plate Bubble Heater 95% air/5% CO 2 Tank Advantages: Simple & portable Easy access to samples Constant pumping-> consistent atmosphere Disadvantages: Requires pre-mixed tank Lots of gas consumed Problem with long term imaging?

8. Design 2: CO 2 Sensor Sample Dish CO 2 Sensor Door Advantages: Sensor -> accurate recording of CO 2 level Do not have to buy a pre-mixed tank Disadvantages: More Expensive Must interface with previously built sensor circuit

9. Design 3: Enclosed Chamber Advantages: Protects microscope Nothing between sample and lens on top side Disadvantages: Requires lots of gas b/c of large volume Harder to maintain constant environment in large volume Not portable -> in the way for people not doing live cell imaging

10. Decision Matrix Possible PointsMixed Air TankCO 2 SensorEnclosed Chamber Ease of construction10 47 Access to samples by user10758 Portability5550 Relative Safety108 9 Cost: Capital Investment10 29 Cost: Operating159 4 Total60494137

11. Future Work Decide on specific parts for design 1 Build new chamber Assemble entire system Testing

12. References 1) De Leeuw, Wim. “Computer at the Microscope: Visualization and Analysis of Three- Dimensional Microscopy Data.” Ercim News Magazine. http://www.ercim.org/publication/Ercim_News/enw60/de_leeuw.html 2) Dailey, Michael E. et al. Nikon Microscopy. http://www.microscopyu.com/articles/livecellimaging/culturechambers.html 3) Nicolas, George (2003) “Confocal Microscope Systems – A Comparison of Technologies” Bioscience Technologies, 11: 12-14. 4) PerkinElmer, Inc. “About Live Cell Imaging” http://las.perkinelmer.com/content/livecellimaging/about.asp 5) W.M. Keck Laboratory for Biological Imaging http://keck.bioimaging.wisc.edu