1. State-of-the-art probes Alan Bigelow Alternative sensing methods Real-time, single-cell analysis techniques

2. 1.Miniature ion-selective single-cell probes Collaboration with the Biocurrents Research Lab at Woods Hole 2.Probe positioner and manipulator 3.Laser excited single-cell optical nanosensors Collaboration with Tuan Vo-Dihn 4.Kambiz Pourrezaei collaboration 1.A Surface-Enhanced Raman Scattering Nano-Needle for Cellular Measurements 2.Carbon Nanotube Cellular Endoscopes 5.Automated Microscope Observation Environment for Biological Analyses (AMOEBA) Outline

3. 1 mm 1m1m 1  m Miniature Ion-Selective Single-Cell Probes These probes are used to study changes of inflows or outflows of small molecules from individual living cells, in response to spatially-defined damage

4. Making Probes

5. Laser-Based Micropipet Pulling Device (Model P-2000; Sutter Industries)

6. Graphite Epoxy Paste Glass Microelectrode O-Phenylenediamine Copper Wire Carbon Fiber Nafion Epoxy The Woods-Hole team have developed sensors for a variety of molecules, such as nitric oxide:

7. Getting these single-cell probes into position, efficiently and reproducibly.... A non-trivial task!

8. Offset Hinge: probe positioning system

9. Other manipulations using the offset hinge mount Cell micro-injection Single cell harvesting Optical fiber based Raman spectroscopy Orientation of medaka embryos

10. Nanobiosensors Collaboration with Tuan Vo-Dinh Advanced Biomedical Science and Technology Group Life Science Division Oak Ridge National Laboratory

11. Nano-biosensor tip Pulled nano-sensors have tip diameters of approximately 40-50 nm Final coated fibers are approximately 200 nm diameter Antibody coated tips for specificity in binding Nanometer diameter tip provides near-field excitation Sensor inside cell

12. Metalic coating of probe end to prevent leakage of the excitation light Gold, Aluminum, or Silver

13. Scanning Electron Microscope Images of a Nanofiber Before Metal Coating (tip diameter ~50nm) After Metal Coating (tip diameter 250-300nm)

15. Nano-probe attachment

24. Automated Microscope Observation Environment for Biological Analyses (AMOEBA)

25. Environment Control User Requests: Physiological conditions Control temperature (e.g. 37 ± 0.5 ºC) Control medium concentrations (CO 2, pH, oxygen, etc.) Initial Solutions: Air-CO 2 mixture: allows accurate particle count; limited time Heater ring: Maintains temperature; cell medium evaporates

26. AMOEBA Flow system for temperature-controlled medium exchange Flexible, user-friendly, modular design offers: Medium aspiration, replacement, and collection Multiple dispensers to change medium type during experiment Additive introduction, such as trypsin to remove cells Sensor insertion to monitor absorbed gas Microfluidics compatibility: Lab-on-a-chip for in-line analysis

27. “Flow” Diagram Example Reservoir I Reservoir II Reservoir III Pump Heater / Cooler Lab-on-a-chip Dispenser Microbeam Dish Hinge mount Additive Inlet

28. Cells were observed for 2 hours with circulating medium at 37 ± 0.5 ºC. Proof of Principle

29. System included heated-window cap, to assist heating control.

30. Lab-in-a-Box Assemble your own system from modules. Automation is computer controlled. AMOEBA is flexible and has potential use in labs across the country and the world. Sensor