In situ video and fluorescence analysis (VFA) of marine particles: applications to phytoplankton ecological studies
Limnol. Oceanogr. Methods 10:807-823 (2012) | DOI: 10.4319/lom.2012.10.807
ABSTRACT: Recent advances in laser and video technologies have enabled single particles to be visualized in aqueous solution. Here, we describe a new instrument enabling the analysis of the fluorescence signatures of marine particles directly in seawater: the Video Fluorescence Analyser (VFA). A field of view is produced inside a measurement chamber by a laser beam at 473 nm that illuminates a shallow region, 3500 μm deep. Individual cells or particles in this field appear as individual spots of light, which are clearly resolved by video against a dark background. The method can resolve particles ranging from 6 μm to several millimeters. The camera is equipped with mobile optical filters: band-pass filter, 520-580 nm, for phycoerythrin visualisation and high-pass filter, >600 nm, for Chlorophyll a pigment. These filters are remotely controlled and displaced in front of the CCD camera, allowing imaging and discrimination between fluorescent particles. We report here experimental procedures and calibrations performed in the laboratory with phytoplankton cells (Dunaliella tertiolecta, Karenia mikimotoi, Pseudonitzschia australis) and calibrated fluorescent beads. Image analysis rocessing enabled particle counts, measurements, and size classification. The auto-fluorescence of individual particles was also tested in situ during a field cruise. In relation to other sensors, the VFA allowed particle enumeration and discrimination and detecting spatial variability of the phytoplankton size spectra in relation to hydrology. The results indicate that fluorescence/video analysis techniques can be easily used in the laboratory or at sea for direct in situ visualization and analyses of phytoplankton populations.