1. Figure 1: Potential sensor behavior; As the CO 2 levels drop in the chamber, the output voltage drops Figure 2: Response of circuit to CO 2 sensor input falling below set threshold Response of Circuit to Changes in CO 2

2. CO 2 Analyzing Circuit Circuit receives voltage input from CO 2 sensor proportional to %C0 2 levels When input voltage drops below threshold (4.75% CO 2 ), the circuit opens valve for a set period of time Knowing volume of box, correct volume of pure CO 2 is added to return CO 2 levels to 5% 20 sec delay in C0 2 sensor calls for valve to remain open at least 20 sec

3. References Bioinstrumentation, John G. Webster (ed.), John Wiley & Sons, 2004 Live Cell Microscopy Environmental Control Systems, www.bioptechs.com, Date accessed 9/26/2006www.bioptechs.com Earth’s Atmosphere, http://en.wikipedia.org/wiki/Earth%27s_atmosphere, Date accessed 10/12/2006http://en.wikipedia.org/wiki/Earth%27s_atmosphere Cell Culture, http://en.wikipedia.org/wiki/Cell_culture, Date accessed 10/1/2006http://en.wikipedia.org/wiki/Cell_culture Special Thanks: Professor John G. Webster, Ph.D., Department of Biomedical Engineering Lance Rodenkirch, Laboratory Manager, Keck Laboratory for Biological Imaging Michael Hendrickson, Graduate Student Burke O’Neal, Department of Biomedical Engineering John Cannon, Department of Chemical

4. Carbon Dioxide Sensor Vaisala GM220 Carbon Dioxide Sensor Measures %CO 2 in a gaseous mixture (air) Capable of measuring up to 10% [CO 2 ] Relays %CO 2 as a voltage Response time is ~20 seconds http://www.vaisala.com/businessareas/instruments/products/carbondioxide/gmm220

5. Bioptechs – FCS3® Maintains accurate thermal control and allows near laminar flow perfusion Controls temp to 50ºC +/- 0.2º Stand-alone temperature controller with an alarm circuit to safeguard cells Micro aqueduct perfusion (see picture at right) Requires an objective heater (not included) Product costs $5000 Existing Products www.bioptechs.com

6. Box: ¼” sheet acrylic used for walls Top of box made of glass, allowing maximum light to pass from condenser Design: Condenser is as close to cells as possible, but easy access to inside of box still available Box dimensions allow for any well on a multi-well Petri dish to be imaged Design incorporates heat pad already owned by client Velcro fasteners ensure tight seal on door Total Volume 9000 cm 3 20 cm 5 cm 10 cm Glass Top Hinged door Velcro fastener Design of Chamber

7. Future Work  Purchase of CO2 sensor and calibrate  Development and integration of solenoid and needle valve system  Integration of additions with existing prototype  Implementation into Laboratory environment  Explore media perfusion possibilities  Refine/improve prototype for large-scale production

8. Live Cell Imaging Two Types Develop studies to establish natural behavior Induced change to study effects of a controlled factor Goal: Imitate cell’s natural environment Nutrient filled serum 5% CO 2 concentration Temperature set at 37° C Saturated humidity Accomplished through: Perfusion CO 2 regulators Heating elements Water pan

9. Design Statement Engineer a cell imaging chamber that maintains an internal 5% carbon dioxide concentration and is adaptable for a future perfusion system.

10. Design Constraints Must not interfere with normal functions of microscope Designed around a welled cell plate Chamber must have internal access Microscope platform 21cm x 25cm x 10cm pH limiting factor, constant 5% CO 2 Humid environment Costs < $1000

11. Abstract This project was designed to create a glass- topped, acrylic imaging chamber that will fit on the stage of a confocal microscope with dimensions 21 cm (l) x 25 cm (w) x 4 cm (h). The chamber is engineered to fit Petri dishes as well as cell plates. The chamber will regulate the internal carbon dioxide to 5% +/- 0.25%, sustain a humid environment and maintain 37 C in the cell culture. The goal of this chamber is to imitate the conditions of cells in vivo.

12. Imaging Chamber Circuit CO2 TankSeries Of Valves CO 2 Sensor Provides Feedback on CO2 Levels Controls CO 2 Levels Overview of Project Design

13. 1)The needle valve reduces the flow rate coming directly from the CO2 Tank 2)Pressure builds in the tube before the solenoid valve 3)The solenoid valve is opened by the circuit 4)The pressurized CO2 between the two valves is released rapidly 5)A constant flow rate, determined by the needle valve, proceeds Bolus (high pressure volume between both valves) High Pressure, High Flow Rate High Pressure, Low Flow Rate Needle Valve Solenoid Valve CO2 Tank On / Off Flow Determined by CO2 Sensor Flow Rate Time Constant Flow rate of needle valve Imaging Chamber Valve Control

14. Reduces the flow rate from the CO2 tank ¼” Pipe, 1 ¾” Long Carbon Steel 5000 psi @400°F $30.33 Solenoid Valve Solenoid directly actuates the valve (no pressure differential required) Designed for low-flow applications 24VDC Compatible with Circuit ½” Conduit Electrical Connection $42.10 Valves Mc Master-Carr 8077K42 Mc Master-Carr 46425K22 Needle Valve

15. Circuit Schematic