Live Cell Imaging ChamberOutlineProblem StatementBackground - NeedBackground – Current TechnologyDesign ConstraintsDesign 1: Mixed Air TankDesign 2: CO2 SensorDesign 3: Enclosed ChamberDecision MatrixFuture WorkReferencesLive Cell Imaging ChamberAndrew Dias, Justin Lundell, Val Maharaj, Jeremy Schaefer, Mike SocieClient: Lance Rodenkirch, UW School of MedicineAdvisor: Prof. Bill MurphyOutline•Problem Statement•Background•Current Technology•Design Constraints•Design Alternatives•Future WorkProblem StatementDesign an imaging chamber that:–Controls gas composition in cell environment–Maintains constant temperature–Is compatible with Nikon Eclipse TE 2000 microscope–Has perfusion capabilitiesBackground - 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)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 controlDesign Constraints•Chamber must:–Maintain 95% air, 5% CO2 atmosphere composition–Maintain temperature of 37.1°C–Fit on 30 cm x 27.6 cm microscope stage–Allow perfusion tubes to access cellsDesign 1: Mixed Air TankImaging ChamberHot plateBubble Heater95% air/5% CO2 TankAdvantages:• Simple & portable• Easy access to samples• Constant pumping-> consistent atmosphereDisadvantages:• Requires pre-mixed tank• Lots of gas consumed• Problem with long term imaging?Design 2: CO2 SensorSample DishCO2 SensorDoorAdvantages:• Sensor -> accurate recording of CO2 level• Do not have to buy a pre-mixed tankDisadvantages:• More Expensive• Must interface with previously built sensor circuitDesign 3: Enclosed ChamberAdvantages:• Protects microscope• Nothing between sample and lens on top sideDisadvantages:• 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 imagingDecision MatrixPossible Points Mixed Air Tank CO2 Sensor Enclosed ChamberEase of construction 10 10 4 7Access to samples by user 10 7 5 8Portability 5 5 5 0Relative Safety 10 8 10 9Cost: Capital Investment 10 10 2 9Cost: Operating 15 9 15 4Total 60 49 41 37Future Work•Decide on specific parts for design 1•Build new chamber•Assemble entire system•TestingReferences1) 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.html2) Dailey, Michael E. et al. Nikon Microscopy. http://www.microscopyu.com/articles/livecellimaging/culturechambers.html3) 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.asp5) W.M. Keck Laboratory for Biological
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