State-of-the-art probes Alan Bigelow Alternative sensing methods Real-time, single-cell analysis...
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State-of-the-art probes
Alan Bigelow
• Alternative sensing methods• Real-time, single-cell analysis techniques
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1. Miniature ion-selective single-cell probesCollaboration with the Biocurrents Research Lab at Woods Hole
2. Probe positioner and manipulator
3. Laser excited single-cell optical nanosensorsCollaboration with Tuan Vo-Dihn
4. Kambiz Pourrezaei collaboration1. 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
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1 mm
1m 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
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Making Probes
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Laser-Based Micropipet Pulling Device (Model P-2000; Sutter Industries)
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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:
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Getting these single-cell probes into position, efficiently and reproducibly....
A non-trivial task!
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Offset Hinge: probe positioning system
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Other manipulations using the offset hinge mount
• Cell micro-injection
• Single cell harvesting
• Optical fiber based Raman spectroscopy
• Orientation of medaka embryos
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Nanobiosensors
Collaboration with Tuan Vo-DinhAdvanced Biomedical Science and Technology Group
Life Science DivisionOak Ridge National Laboratory
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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
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Metalic coating of probe end to prevent leakage of the excitation light
Gold,Aluminum,or Silver
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Scanning Electron Microscope Imagesof a Nanofiber
Before Metal Coating(tip diameter ~50nm)
After Metal Coating(tip diameter 250-300nm)
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Nano-probe attachment
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Automated Microscope Observation Environment for Biological Analyses
(AMOEBA)
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Environment Control
User Requests: Physiological conditionsControl temperature (e.g. 37 ± 0.5 ºC)
Control medium concentrations (CO2, pH, oxygen, etc.)
Initial Solutions:• Air-CO2 mixture: allows accurate particle count; limited time
• Heater ring: Maintains temperature; cell medium evaporates
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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
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“Flow” Diagram Example
Reservoir I
Reservoir II
Reservoir III
PumpHeater / Cooler
Lab-on-a-chip
Dispenser
Microbeam Dish
Hinge mount
AdditiveInlet
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Cells were observed for 2 hours with circulating medium at 37 ± 0.5 ºC.
Proof of Principle
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Proof of Principle
System included heated-window cap, to assist heating control.
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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