Presentation on theme: "Development of a Portable Fluorescence Bacterial Detector Texas A&M- Commerce."— Presentation transcript:
Development of a Portable Fluorescence Bacterial Detector Texas A&M- Commerce
People Team Members – David Andrew Jacob – Will Negrete – Jeff E. Landry – Holly Pryor Faculty Advisor – Dr. Frank Miskevich
Why is monitoring important to people both on earth and in space? on earth and in space?
Introduction Microorganisms can be found almost anywhere on earth. There are more microorganisms living in and on a human than the sum of the cells that make up that human. Some are dangerous to humans, others are benign.
Introduction Bacteria are a major contributor to human disease Fast generation time (exponential growth) Can spread quickly in compact populations as seen in space stations and space craft
Monitoring Critical in Space Air and Water Recycled Limited Personal Hygiene Infectious Disease spreads quickly in close living quarters Difficult to isolate sick individual from crew Despite our best efforts microbes still inhabit the space station Fungus Growing on Wall of ISS
Our Method Culture Independent Bacteria marked with a non-toxic, fluorescent DNA binding dye (Hoechst 33258) Each fluorescing bacteria is counted to give X bacterial fluorescent units (BFUs) Bacterial Fluorescent Units Test photo from microscope. Note: artifacts are not bacteria, nor should “cloudy” areas exist.
Our Method Counts both dead and alive bacteria Does not require prior knowledge of organism to be cultured to quantify Estimated that only 1% of present bacteria grow in culture dependent bacteria (La Duc, 2003) Bacterial Fluorescent Units
Proof of Concept Work done by Joseph Harvey, M.S. BFU results generated from our method correlates (P=0.8051) to flow cytometer results Flow Cytometer results pictured above. Shows both dead and alive bacteria.
Escherichia coli suspensions used to test device – Gram-negative rod, Non-sporulating – 2 μm long X 0.5 μm in diameter – Cell volume = ~0.6 - 0.7 μm 3 – Very common flora in human GI tract
Sample Preparation Hoechst 33258 is added to liquid bacteria sample at 1 micro liter per milliliter sample Liquid sample is then drawn up into syringe Sample is pass through 0.2 micron filter Filter is put into sample holder and photographed
Sample Holder Polycarbonate Filter Sandwiched between parts B and C (Above & Right) Parts A and D attached to stepper motor. Allows parts B & C to be held in front of the camera assembly
Detector Overview 1. Digital Camera 2. Infinitube 3. UV LED 4. Bandpass filter 5. Microscope objective lens 6. Stepper motor 7. Laptop 8. 19.2 VDC Power supply 9. Motor driver 10. Laptop Interface 11. Dichroic mirror
Filters Dichroic lens reflects 350nm light and allows 450nm sample emission to pass through 450nm bandpass filter selects for light very close to the 450nm spectrum “cleans up” picture seen by camera by reducing noise
Integration of Parts Stepper motor and UV LED activation coordinated by programmable step motor controller Relay Used to allow 5 VDC TTL activation of UV LED Single USB hook up to laptop controller Note Addition on Solenoid and controller board; Triggered from PSMC
Software Stepper motor controller program Nikon D80 camera software IMAGEJ Counting Macro Major Problem Solved: Computer Science Graduate Student Joining Team Next Semester
IMAGEJ Free software by National Institute of Health (NIH) Raw Images sharpened Delineates boundaries positive for bacteria and background Counting macro used to count bacteria Clusters of bacteria counted based on area and individual number of bacteria estimated bacterial image selected areas
The Detector Current Work: Integrate camera trigger and stepper controller Integrate camera trigger and stepper controller Increase UV light intensity Increase UV light intensity Increase structural integrity & refinement of device Increase structural integrity & refinement of device
Increase UV Intensity Light generated by UV LED(s). Reflected off dichroic lens towards sample or generated by “ring of LEDs” near sample. Ring of LEDs added to increase light intensity. Single LED source from microscope tube proved to be inadequate. Both sources are going to be used in future. Activated on same circuit as original LED.
Increase UV Intensity Five UV LEDs in series for ~19.2V draw from battery. LEDs will be focused so that their beams converge on the same point within the focal plane of the camera.
Camera Trigger Trigger activated via stepper motor controller
Camera Trigger Force limited by solenoid controller board so as not to damage trigger Operated off 19.2VDC battery activated by 5VDC TTL signal
Strengthening of Device Structure Must be rigid otherwise focus changes are possible. Focal length isvery small. “L” brackets added.
Strengthening of Device Structure Motor shim added to assist in maintaining coplanar focus. Critical to function and ability of get clear, uniformly focused pictures.
Integrate all software (camera controller, motor / LED controller, IMAGEJ and counting macro) into one easy to use package that can be loaded onto the detectors memory stick and allow USB “Plug & Play” compatibility Integrate all software (camera controller, motor / LED controller, IMAGEJ and counting macro) into one easy to use package that can be loaded onto the detectors memory stick and allow USB “Plug & Play” compatibility Graduate computer science student Recruited to assist with integration of Software components into single, user-friendly package.
White Blood Cell Counts Erythrocytes (Red Blood Cells) are anucleated. White blood cells have nuclear material. Left: Electron micrograph of RBC Above: stained in purple, WBC (neutrophil)
Our dye (Hoechst 33258) stains only DNA. Therefore, we can select preferentially for WBC and utilize the same process to estimate number of WBCs present in a given volume on blood. White Blood Cell Counts
Method of operation very similar. Method of operation very similar. Given a specific volume of blood our detector can generate WBCs per volume data. Given a specific volume of blood our detector can generate WBCs per volume data. White blood cell counts good marker for immune function and disease states. White blood cell counts good marker for immune function and disease states. White Blood Cell Counts
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