About in situ Flow Cytometry

Intercalibration = instruments presentation

thyssen — 2008-05-13T16:36:46Z

The Cytosub flow cytometers (Cytobuoy b.v.) are able to analyse large phytoplanktonic cells (1 to 1000 µm and a few mm in length, but a specific optical unit (picoplankton option) can look at < 1 µm ) and relatively large water volumes (up to 4 ml per sample).

The fluidic, optics and electronic are modified in order to resolve the low concentrated and highly diversified phytoplankton cells in natural waters. They are capable for operating on line or as a submersible instrument. The Cytosubs can be deployed down to 200 m depth helpful to a cylindrical aluminium covering. The seawater was pumped to fill a sample loop of 4 ml before entering the flow cell in order to avoid external turbulences and operate at atmospheric pressure. Minimal sampling frequency is approx. one sample every 5 minute corresponding to the flushing of the sample loop and the data transfer, depending on the concentration and on the size of the cells.

LMGEM's Cytosub had an external power source (24V AC) coming from an on land desk through a waterproof data-power cable. This cable allowed the sending of the analysed sample after each run. The sample flow was controlled by a peristaltic pump working at 7.8 µl.s-1 during the temporal study in the Bay of Marseille. It flowed at 8.3 µl.s-1 ¬during the intercalibration test with the NOCS's Cytosub because the sample tubing was replaced. The sheath fluid flowed at a rate of 80 ml.min-1.

Each particle was intercepted by the solid-state laser beam (Coherent Saphyre, 488 nm, 15 mW). The side scatters and fluorescence signals were dispersed by a concave holographic grating and collected via a HPMT (Hybrid Photomultiplier). The forward scatter signal was collected via a PIN photodiode. The red (FLR), orange (FLO) and yellow (FLY) fluorescences were collected in the wavelength ranges 734-668, 601-668, 536-601 respectively. Data recording was triggered by the forward scatter signal. The shape of the signals was encoded with a frequency of 4 MHz and data were saved in distinct 64 kbits grabbers. Particles flew at a rate of 2 m.s-1 through the 5 µm laser beam so that for instance the signal shapes of 1 µm beads would be defined by 12 points. The sheath fluid and the seawater sample were mixed together at the exit of the flow cell and filtered onto 0.2 µm nucleopore filters in order to be recycled.

NOCS's Cytosub was similar to the LMGEM one except that power source came from lithium battery (24V DC) encapsulated in an external waterproof and pressurised cylinder. Further more, the analysed samples were saved in a data logger and communication (data transfer and sampling programming) to a computer used a BlueTooth connection. Sample flow reached 11 µl.s-1.

PUBLICATION UPDATES

2008-05-13T16:29:29Z

Publication :

Melilotus Thyssen, Beatriz Beker, Dilek Ediger, Doruk Yilmaz, Nicole Garcia, Michel Denis. In situ flow cytometry coupled with conventional methods : how dot hey fit ?. JMBE. Soumis.

Melilotus Thyssen, Nicole Garcia, Michel Denis. Sub meso scale phytoplankton distribution in the north east Atlantic surface waters determined with an automated flow cytometer. Biogeoscience. Soumis.

Melilotus Thyssen, Delphine Mathieu, Nicole Garcia, Michel Denis. Short-term variation of the phytoplankton assemblage in the bay of Marseille (France) monitored by in situ flow cytometry. Journal of Plankton Research. En révision.

Hera Karayanni, Urania Christaki, France Van Wambeke, Melilotus Thyssen, Michel Denis (2008). Heterotrophic nanoflagellate and ciliate bacterivorous activity and growth in oceanic waters : a seasonal study at mesoscale in NE Atlantic. Aquatic Microbial Ecology. Sous presse.

Melilotus Thyssen, Glen A. Tarran, Mikhail V. Zubkov, Ross J. Holland, Gérald Grégori, Peter H. Burkill and Michel Denis. (2008). The emergence of automated high frequency flow cytometry : revealing temporal and spatial phytoplankton variability. Journal of Plankton Research, 30 : 333-343 ; doi:10.1093/plankt/fbn005.

Camila Fernandez Ibanez, Thyssen Melilotus, Michel Denis. (2007). Microbial community structure along 18°W (39°N - 44.5°N) in the NE Atlantic in late summer 2001 (POMME programme). Journal of Marine Systems. 71(1-2) : 46-62.

Samer Sakr, Melilotus Thyssen, Michel Denis, and Cheng-Cai Zang. (2006). Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120. Journal of Bacteriology. 188 : 5958-5965.

Gaëtan Gogniat, Melilotus Thyssen, Michel Denis, Cesar Pulgarin, Sam Dukan. (2006). The bactericidal effect of TiO2 photocatalysis involves adsorption onto catalyst and the loss of membrane integrity. FEMS Microbiology Letters. 258:1-18.

Melilotus Thyssen, Dominique Lefèvre, Guy Caniaux, Josephine Ras, Camila Fernandez Ibanez, Michel Denis (2005). Spatial distribution of heterotrophic bacteria in the North East Atlantic (POMME study area) during Spring. Journal of Geophysical Research, 110(C7), C07S1.

Anne Maixandeau, Dominique Lefèvre, Hera Karayanni, Urania Christaki, France Van Wambeke, Melilotus Thyssen, Michel Denis, Camila Fernández I., Julia Uitz, Karine Leblanc and Bernard Quéguiner. (2005). Seasonal and mesoscale variability of Gross Community Production and Respiration in relation to microbial food web structure in the Northeastern Atlantic Ocean. Journal of Geophysical Research., Vol. 110, No. C7, C07S17.

Talks :

Principal topic of the talks = “in situ flow cytometry and phytoplankton ecology”

— IMBER/LOICZ meeting. Shangai. 17-21 September 2007. Award of the Young Scientist best presentation.

— Laboratory of “Ecosystèmes Lagunaires” Montpellier. 25 January 2007. Seminary.

— Colloque of « Sciences et Technologies Marines du futur ». 19 and 20 May 2005. Marseille.

— Symposium on Single cell analysis of planktonic microbes. 18-19 October 2005. Banyuls sur Mer.

— Atelier Expérimentation et Instrumentation (AEI). 31 January-1st February 2006. Brest.

— Conférence Internationale des Limnologues d'Expression Francaise (CILEF).17-21 March 2006. Tunisia.

— International Society of Analytical Cytology (ISAC). 20-24 May 2006. Quebec.

— ISMER Rimousky May 2006. Seminary.

— INRA Thonon les Bains September 2006. Seminary.

— Congrés de l'Association Française de Cytometrie. Invited. 18-19 October 2006. Bruges.

— Congrés annuel de la Deutsche Geselischaft für Zytometrie e.V. Invited- Plenary session. 19-22 October 2006. Leipzig.

— Congrés Méditerranéen d'Océanologie. 20-22 November 2006. Alger.

Diverse articles :

Michel Denis, Melilotus Thyssen. 2007. Quand océanologie et réinsertion sociale se concertent. Nouvelles Scientifiques. Journal de l'INSU. http://www.insu.cnrs.fr/a2230,quand-oceanologie-reinsertion-sociale-se-concertent.html

Melilotus Thyssen and Michel Denis. 2007. High-resolution temporal and spatial sampling of phytoplankton. IMBER Update. http://www.com.univ-mrs.fr/PRECYM/IMG/pdf/IMBER_Update_Oct07.pdf

Michel Denis, Melilotus Thyssen. 2007. Océanologie et jeunes en difficulté. La lettre du Grand Luminy. 2007. http://www.agl.univ-mrs.fr/LettreGL/Article.php ?num=984

Luis Felipe Artigas, Ivaneide do Rosario Marinho – Jaussaud, Jean-François Ternon, Melilotus Thyssen, Melika Baklouti, Beatriz Beker and Daniel Guiral. 2008. “Bacterioplankton dynamics and ecological role in estuarine, coastal and shelf waters of French Guiana”. VII Ecolab Workshop. Maapa, Brasil.

ACYPHAR

thyssen — 2007-05-25T16:55:24Z

Two researchers (Melilotus Thyssen and Michel Denis) from the « Laboratoire de Microbiologie, Géochimie et Ecologie Marines, Centre d'Océanologie de Marseille » (LMGEM-COM, France), combined a scientific project with a program to rehabilitate juvenile delinquents. DEFERLANTE, an association under contract with social and judicial services, uses the ship Fetia Ura, a 33 m schooner (belonging to SARL SEANERGIES OCEANES) to help young guyes with family and school dropouts regain self confidence and build social connections through high-sea sailing.

The two researchers joined the Fetia Ura (a crew of 5 people, 2 instructors and 6 teenagers) at Horta (Faial, Azores Island) on its return from French Guyana. From 14 to 23 April 2007, the ACYPHAR (Atlantic flow CYtometry spatial study of PHytoplankton through Automated Recording) scientists studied the surface phytoplankton (< 50 μm) distribution between Horta and Lorient (France). A CytoSub (Cytobuoy) automated flow cytometer was used to analyse seawater pumped into a buffering reservoir every 15 min, which corresponded to a distance interval of about 2 nautical miles. In parallel, the seawater properties were determined with an autonomous CTD (SBE 37SMP).

These high frequency measurements were collected to allow better characterization of the spatial heterogeneity of the oceanic phytoplankton distribution.

The scientists contributed to the rehabilitation program of DEFERLANTE by providing the teenagers and their instructors basic information on the ocean and the marine ecosystem. This took place through discussions and interactive Powerpoint presentations. An epifluorescence microscope coupled to a CCD camera (CYSCOPE, Partec) was used to display on a computer screen organisms collected with a phytoplankton net. The teenagers were also made responsible of some subsampling scheduled every 4 hours. Through the interaction with the scientists, the teenagers acquired a high interest for the marine environment, the global change and the scientific rigorousness. They thus became aware of the importance of marine phytoplankton as regulators of greenhouse gases and as the basis of the oceanic food chain.

The ACYPHAR cruise demonstrated the feasibility and cost-effectiveness of automated flow cytometry analysis of phytoplankton on board marine vessels. This original combination program provides the opportunity to collect valuable data that has been heretofore out of reach.

The sampling structure inside of the boat.

The CTD was dropped into the black garbage where the pumped sea water came out from the Cytosub sampling structure.

The young teens were sampling water every 4 hours in order to analyse the nutrient contents later on.

pictures : Michel Denis 2007

Technical competences

thyssen — 2007-02-19T13:45:06Z

Technical competence :

— Bench top flow cytometry analysis (instrument : Cytoron Absolute from Ortho Diagnostic Systems) :

marine heterotrophic and autotrophic planktonic cells, E.Coli cultures.

— Sample preparation (fixation, staining, fluorescent beads, cryoconservation).

— In situ flow cytometry handling and data analysis (instrument : CytoSub – CytoSense from CytoBuoy, b.v., NL)

— CTD SBE 37 handling

— EcoWetLab fluorometer handling

— Dissolved oxygen measurement using the colorimetric Winkler method, modified by Carpenter (1965), Caritt & Carpenter (1966) & Culberson (1991).

— Computer coding knowledge (C, C# ; VB, SQL, BASH, R)

Software :

— WINLIST of Verity Software (Cluster analysis), WinMDI

— OCEAN DATA VIEW from R. Schlitzer, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany

— R, Word, Excel, Access, Power point, Photoshop…

Temporal study - photo galerie

thyssen — 2007-02-19T09:48:47Z

JULY 2005

The Cytosub of the LMGEM was put in Michel Denis's car.

And sent to the Roucas Blanc harbour (nice area for summer sampling)

The Cytosub was fixed on a stainless steel armature and directly connected via a data/power cable to a computer approximatly 200 m away.

For the first ideployment, a team of strong man gave their help.

Seeing the instrument under the water is allways a very strange feeling.

My super assistant, Delphine Mathieu. She was there for a part of the sampling period and did a fantastic job, whilst I sat and posed for photographs (below).

During the August's holiday period, Michel and I had to find out the best way to bring the instrument into the water.

Yealm aventure - photo galerie

2007-02-19T09:48:10Z

The Cytosub flow cytometer involved in this adventure is at the Plymouth Marine Laboratory (PML) and is used by Dr Glen Tarran. This is the prototype CytoSub and was developed through a UK Natural Environment Research Council (NERC) Autosub Special Topic Award between Prof. Peter Burkill at PML and Dr. Alex Cunningham at the University of Strathclyde. The project was called SSCAPA – Sub-surface Cell and Particle Analysis (Project # GST/02/2153) and ran between 1998 and 2001.

The opportunity of mooring the Cytosub in the river Yealm was made possible by local oyster farmers Martin Oates and Steve Allen. They kindly offered use of their boat to transport the CytoSub to a mooring buoy in the Yealm estuary.

First, oysters had to be collected.

Cytosub was cheked,

prepared for deployment,

and fixed to a buoy in the middle of the estuary for 48 hours.

During this time, we took seawater samples using a small inflatable boat in order to measure temperature and salinity variations and also preserved samples to quantify phytoplankton and bacteria using conventional flow cytometry.

Of course, we learned to injoy tea time...

Intercalibration photo galerie

thyssen — 2007-02-19T09:47:39Z

December 2006

Sampling sites

Location boat, departure in the morning

Instruments fixation

Here we go !!

Instrument programming and procedure checking

Glen Tarran and Ross Holland enjoyed the sampling

View of Notre Dame de la Garde on the way back