Biofouling and Ballast Water Management Conference. National Institute of Oceanography Goa, India 5,6 & 7 February, 2008

Transcription

1 Biofouling and Ballast Water Management Conference National Institute of Oceanography Goa, India 5,6 & 7 February,

2 FlowCAM Technology- The Integrated System for Ballast Water Analysis and Regulatory Compliance Kent Peterson, C.E.O. Fluid Imaging Technologies, Inc. Yarmouth, Maine, USA

3 Agenda Introduction FlowCAM Technology Demonstration Examples Summary

4 Fluid Imaging Technologies Founded 1999 Maine, USA (BLOS) Flow Cytometer And Microscope (FlowCAM) > 100 FlowCAMs sold Product Development Depth-of-Focus Technology VisualSpreadsheet 2.0

5 Bench Top FlowCAM

6 Portable FlowCAM

7 FlowCAM Proprietary Technology Camera Flow Cytometer FlowCAM Microscope

8 Patented Features Depth-of-Focus Enhancer Lens S Interactive Scattergram Software

9 FlowCAM Methodology

10 FlowCAM Specifications Camera High Resolution 1024 (H) x 768 (v), Digital FireWire (.256 µm/pixel) Progressive Scan CCD Black & White or Color Computer Intel Core 2 Duo 1.66 GHz, 2 GB RAM, 80 GB Hard Drive, Windows XP Pro Laser 25 mw, 488 nm Solid-State Blue Laser or 532 nm Green Laser

11 FlowCAM Specifications Particle sizes Imaging 3 µm to 3 mm Detection.5 µm to 3 mm Objectives j 2x, 4x, 10x, 20x Flow Cell 50 µm to 2 mm Processing Capability Flow -.25 ml/min to 12 ml/min Density - 50,000 particles/ml (Auto Trigger)

12 FlowCAM Users Scripps Inst of Oceanography U.S. Naval Research Labs Rensselaer Polytechnic Inst National Oceanographic Center Virginia i i Inst for Marine Sciences U.S. EPA Alfred-Wegner Inst. United Nations University Oceanographic Institute of China Royal Netherlands Institute for Ocean Science Korean Oceanographic Research & Development Inst. (KORDI) Texas A&M University Harbor Branch Oceanographic Inst. Australian Inst. for Marine Sciences University of Hawaii University of Munich Plymouth Marine Lab U. of Hiroshima Louisiana State University Battelle Marine Labs

13 28 FlowCAM Image Parameters Morphological Parameters Length Width ESD ABD Aspect Ratio Elongation Perimeter Particles per Chain Relative Chlorophyll Relative Phycoerythrin Scatter Signal Value Image Parameters Transparency Intensity Sigma Intensity Compactness Roughness Average Red Average Green Average Blue Red/Green Ratio Blue/Green Ratio Red Blue Ratio

14 New Technology- Digital Signal Processing (DSP) 1 board replaces 7 3 fluorescence channels, 1 scatter detection channel 600MHz (30 times faster than 7 boards) Smaller particle analysis capability Faster throughput Finer image quality

15 Interactive Scattergram -.lst file Source - U.S. EPA

16 FlowCAM Data Acquisition

17

18 NEW: FlowCAM Color Images

19 FlowCAM Images-.tif files

20 FlowCAM Data -.fcm files Particle Time of Ev File Numb Cell Area Channel 1 Channel 1 Particles p FFT Area Channel 2 Channel 2 feret_max feret_min_cell X Cell Y Cell ESD E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E

21 Particle Library

22 Create Filter Select Images from sample to base Filter on, create or use Library, or select parameter value(s) Test Filter (select again, if deemed necessary) Utilize Scoring Dialog to improve Filter o Adjust Parameter Field Values o Start with Filter Score

23 Filter Dialog

24 x Automated Classification Creating Classification Determine taxonomic groups of Interest Link existing Filters to build Template Utilize existing Templates Run Automated Classification o Inspect Classifications o Looked for Missed Images (using individual Filters)

25 Lake Kinneret, Israel Sample

26 x x Automated Recognition Accuracy Lake Kinneret Organism Run # Class d # Mis-Class % Accurate # Missed % Missed Cladoceran % 0 0% % 1 25% Copepod % 0 0% % 0 0% Egg % 0 0% % 0 0% Diatom Chain % 2 100% % 1 11% Average 8 99% 17%

27 File Processing Mode 27

28 File Reprocessing 28

29 FlowCAM Ballast Water Analysis System Per Liter Live Particles P blue line indicates 10 viable organisms per liter

30 FlowCAM Applications Ballast Water Analysis Oil-in-Water Analysis Wastewater treatment Algal l Bloom Warnings Ocean Transects Ferry Box Zebra Mussel Birefringence

31 Biocide Treatment- Before Untreated Ballast Water Cape May July 12 ' Pennate diatoms 1118 Particles per ml Ciliates and dinoflagellates Log Chlorophyll Ciliates from Cape May untreated water Particle Size (um) X2.5 Prorocentrum sp. From Cape May untreated water

32 Biocide Treatment- After Log Chlorophyll ppm Degussa AG PERACLEAN OCEAN Day 2 Cape May July 12 '01 Pennate diatoms Ciliates and dinoflagellates 182 Particles per ml Particle Size (um) X 2.5 FlowCAM images of 10 to 12 um particles in Cape May water treated with 100 ppm Degussa AG- PERACLEAN OCEAN

33 Sonic Treatment Results Before After

34 Algal Cell Presence Phytoplankton >12um analyzed and counted FlowCAM measured 50% reduction with Sonic FlowCAM substantiated sonic & ozone success

35 Using Metabolic Probes to Determine Cell Viability using FlowCAM Viability Probes - Examples fluorescein diacetate (FDA) 5-carboxyfluoroscein diacetate (CFDA) Viability probe testing - FlowCAM - Used Phytoplankton cultures (e. g. Heterocapsa Dinoflagellate) Tested 1 FDA uptake using Preserved culture (0.5% formaldehyde) Tested 2 FDA uptake using Live culture Test 1 Preserved unstained + FDA Test 2 Live stained fluoresces green with blue excitation once incorporated in the cell

36 Cell Viability Analysis (Heterocapsa) live dead FDA stained LIVE TEST FDA stained DEAD TEST

37 NRL Developed Dyes and Image Processing/Classification Algorithms Automated Tetraselmis Live / Dead Classification Using FlowCAM FlowCAM Used in Conjunction With NRL Developed Dyes and Image Processing/Classification Algorithms Is Currently Being Validated as a Means to Analyze The Complex Mixed Samples Generated During Production Testing at the NRL Ballast Water Treatment Facility in Key West and Determine: The Concentration of Each Phytoplankton Surrogate Species The Concentration of Indigenous Phytoplankton of Various Classes The Viable Cell Percentage for Each Phytoplankton Species Counted

38 NRL Developed Dyes and Image Processing / Classification Algorithms Automated Tetraselmis Live/Dead Classification Using FlowCAM Red Histogram Live Tetraselmis Cells Blue Histogram Dyed Dead Tetraselmis Cells Blue Laser Florescence Channel Affords Live / Dead Cell Classification

39 Comparison Of FlowCAM Counts To Manual Hand Counts Samples Were Homogeneous Diluted Thalassioira Weissflogii Stock Solutions 2.5E E+06 of TW/ ml Mean # 1.5E E E E+00 Manual Hand counts Flow CAM Total Counts using all 3 Channel (Experiment A) These Tests Were Performed By The Naval Research Laboratory In Support Of Their Ballast Water Treatment Test Facility in Key West, Florida

40 FlowCAM Viability using FDA and Artemia

41 FlowCAM Viability Analysis (Artemia) FDA Stained LIVE FDA Stained DEAD

42 FlowCAM Viability Analysis Using Artemia Channel 2 Peak Fluorescence Stained Artemia LIVE Channel 2 Peak Fluorescence Stained Artemia DEAD

43 Cell Mortality using Sytox Green DNA stain Sytox Green enters cells with compromised cell membranes only (non-viable) Dead cells Dead cells Live cells Dead Heterocapsa stained with Sytox Green Sytox stained field sample

44 Zooplankton Viability using Neutral Red For cells greater than 50um the use of the Neutral Red viability stain could be used in combination with a color FlowCAM. Live copecods from a NR stained sample Zooplankton stained red are metabolically active (viable) and cells that are unstained are not viable. Dead copecods from a NR stained sample

45 Zooplankton Viability using Neutral Red Other examples of Neutral Red stained cells (viable cells) from a natural concentrated field sample FlowCAM has the ability to sort cells based on any image parameter including color (average red intensity) Viable Neutral Red stained Dead

46 Boothbay Harbor, Maine Zooplankton sample Live vs Dead/preserved using Neutral Red Staining LIVE nauplii DEAD nauplii

47 Boothbay Harbor, Maine Neutral Red Staining MIX 50:50 Analyzed data using a two parameter Filter (Average Red & Ratio Red Blue)

48 Boothbay Harbor, Maine Neutral Red Staining Initial Data Results using Color Filtering Sample Type Total Count Zooplankton Concentration % LIVE using color filter LIVE cells/ml 84% LIVE DEAD cells/ml 15% MIX 50: cells/ml 51% DEAD

49 FlowCAM Ballast Water Analysis System (pass)

50 FlowCAM Ballast Water Analysis System (fail)

51 Summary FlowCAM H/W & S/W Technology Application Versatility Productivity it Enhancement BWTS Validation & Monitoring

52 Contact Us Kent Peterson, C.E.O. Fluid Imaging Technologies, Inc. Yarmouth, Maine USA