Progress Report

CICEET Progress Report for the period 9/16/06 Through 3/15/07

Project Title: Improved Characterization of Microalgal Abundance and Taxonomic Status through Laser-Induced Fluorescence (LIF)
Principal Investigator(s): Hugh MacIntyre (Dauphin Island Sea Lab) and Richard Cox (Kaitech Inc.)
Project Start Date: 9/1/06

Project Objectives for This Reporting Period
Objectives
The objectives of the 1st and 2nd quarters in Y1 were to construct a 2nd generation laserinduced fluorescence (LIF) instrument and to conduct electro-optical and sensor performance testing.

Tasks to meet objectives
Perform design, analysis, and modeling of fluorescent signals
Prototype the laser alignment mechanism and demonstrate its functionality
Prototype the fluorescence sensor assembly for laboratory testing

Progress on Tasks
Design and Analysis. Several optical designs for the fluorescence sensor were modeled using ZEMAX software to tradeoff possible laser beam size and laser beam divergence requirements for effective fluorescence detection. The drivers were designed to maximize fluorescence power density at the detection position resulted in selecting collimated designs for both the laser and for the detection optical paths.

Demonstrate Laser Alignment Mechanism. To evaluate the detection of the scattering and fluorescence signals completed above three different test fixtures were fabricated. Fixtures 1 & 2 had fixed mechanical displacements and orientations between the laser beam and the detection optics and fixture 3 provided x & y displacement capabilities to access the variability of these requirements. Tests using Maalox as the scattering agent established the sensor's physical design requirements.

Prototype of the Fluorescence Sensor Assembly. A design incorporating multiple lasers for water sample excitation and orthogonal single point fluorescence emission detection was developed that permits up to five different lasers to be used for sample excitation. Unique features of this design include reflectors to augment and enhance the fluorescence signal. Individual part drawings and specifications were developed and the parts have been procured. At this time the opto-mechanical hardware is in hand, initial LabView software to operate and control the hardware has been developed and is available, lasers are due early February, and we plan to begin laboratory testing of the new fluorescence sensor by March 1, 2007.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
No

Dissemination activities during this reporting period
A synopsis of the project’s aims were published in a Dauphin Island Sea Lab press release (http://press.disl.org/10_12_06_hugh.html), 10/12/06. In response, a description of the project was published by Frost and Sullivan’s on-line Technical Intelligence report (http://www.ti.frost.com), 12/15/07.

Following the PI workshop in Mobile, 1/17/07, issues of intellectual property rights, public disclosure and patent protection were discussed with Andy Helminger, RTI.

Difficulties
None

Data Generated to date
Documentation of the engineering analysis completed to date has yet to be compiled. However, current efforts are focused to deliver an operational sensor to DISL for testing and evaluation. Once the sensor's testing and evaluation is accomplished a technical report can be prepared.

Project Objectives for Next Reporting Period

Objectives
Sensor performance testing
Consensus signature definition
Prototype absorption/turbidity correction assembly for laboratory testing

Work plan to Meet Objectives
The central activity planned for the next reporting period is for Kaitech to deliver a laboratory LIF to DISL so that its responses/capabilities can be tested on a library of microalgal cultures. We anticipate that the equipment will be delivered to DISL by mid- March.

Sensor performance testing. The instrument’s limit of detection, S:N ratios and range of linearity will be evaluated at each excitation wavelength using the dye Basic Blue 3 and representative cultures of microalgae (i.e. a cyanobacterium, chlorophyte, cryptophyte and chromophyte).

Consensus signature definition. Consensus signatures will be derived by measurement of the responses of 5-6 clones each of representative cyanobacteria, chlorophytes, cryptophytes and chromophytes (including diatoms, dinoflagellates, prymnesiophytes and pelagophytes).

Prototype absorption/turbidity correction assembly for laboratory testing. Approaches to determining signal attenuation due to background absorption and scattering by interfering chromophores (chromophoric dissolved organic matter and detritus) in natural samples were discussed by the PIs at a meeting at DISL, 1/25/07. The physical constraints of the laser and detector spatial arrangement preclude integrating this within the interrogation chamber. The agreed-upon approach is for a second-stage detector whose optical path parallels the fluorescence interrogation chamber’s. Kaitech's two-point attenuation sensor design will be adapted to provide this.

Dissemination Objectives for next reporting period
The results of the 1st-generation instrument will be presented by MacIntyre in an invited talk at the AquaFluo conference (http://www.aquafluo.cz/program.html) in Nove Hradny, the Czech Republic, May 27 June 1, 2007. If feasible, the 2nd-generation instrument will be demonstrated in an accompanying workshop, led by MacIntyre and Dr Tammi Richardson (University of South Carolina).

The principles and preliminary data will be incorporated into a 2-week, 2-credit graduate course developed and taught by MacIntyre at DISL, “MAS512, Chlorophyll Fluorescence Techniques”. If feasible, the 2nd-generation instrument will be tested in the accompanying laboratory exercises.

Overall Project Timeline Update
Work on the project is proceeding on schedule with the proposed time-line.

Expenditures
Expenditures are in the range anticipated for the work accomplished to date.

End User Advisor Feedback
End User Advisor: Dr. Erik M. Smith
Organization: University of South Carolina, Baruch Marine Field Laboratory & North
Inlet Winyah Bay National Estuarine Research Reserve
Location: Georgetown, South Carolina
Phone number: (843) 546-3623
E-mail: erik@belle.baruch.sc.edu

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
If this research is successful, the proposed LIF technology will greatly improve our ability to continuously monitor and detect change in phytoplankton biomass and pigment composition. This would have tremendous application in generating the data necessary for increasing our fundamental understanding of phytoplankton variability, as well as our ability to detect and respond to the onset of potentially harmful algal blooms. At this stage, documentation of the engineering analysis completed to date has yet to be compiled by the PI. However, from the current progress report, it appears clear that the PIs have made significant progress in the development of the prototype laser alignment and fluorescence sensor assemblies, and that the project is proceeding on schedule.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
The key challenges to application of this technology appear to me to be strictly technological: Will this actually provide quantitative measures of phytoplankton biomass and pigment composition in the field, and will application of the technology be costprohibitive to routine inclusion in research and monitoring programs? At this point in the project, it is simply too early to tell. If successful, however, both the basic research and the resource management communities should eagerly embrace the use of the technology.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
N/A.

Questions/comments/ suggestions for the researchers?
None. All appears on schedule, and I look forward to seeing the results of the coming laboratory testing of the LIF prototype.

End User Advisor: Mr Mark Trice
Organization: Maryland Department of Natural Resources
Location: Annapolis, MD
Phone number: (410) 260 8DNR
E-mail: MTrice@dnr.state.md.us

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?

Questions/comments/ suggestions for the researchers?
I have no comments to provide for your progress report since you are still in the technical fabrication of the instrument, which is obviously not my area of expertise

End User Advisor: Dr Robert Nuzzi
Organization: Suffolk County Department of Health Services
Location: Yaphank, NY
Phone number: (831) 852 5700
E-mail: robert.nuzzi@suffolkcountyny.gov

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.

The potential applications remain as indicated previously. Presently employed methods for identifying cell types and estimating population size rely on laborious microscopic analysis rarely undertaken because of expense. This is especially true in case of nano- and picoplankton. At present, this agency only investigates plankton populations when they are in bloom condition, garnering little information on population dynamics. We do, however, have considerable data on water quality trends, and the availability of a reliable automated methodology for plankton analysis would provide invaluable data, and would be a major step forward in the study and understanding of aquatic ecosystems. The possibility of in-situ sensors that could replace manual sampling, and provide early warning of bloom development, is especially interesting.

An immediate application would be for consistent monitoring of the red tide organism Alexandrium fundyense, especially as it appears to be migrating southward from New England

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
The technology must be reliable, affordable and easy enough to use for routine monitoring of plankton populations. The investigators recognize the challenge associated with interfering particles and chromophores, which I suspect will be particularly significant in estuarine waters, especially those closely associated with humic and/or organic runoff.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?

PI Response to End User Advisor Feedback
The project is at too early a stage to predict either reliability or cost, as noted by Dr. Smith.