Through-Focus Scanning Optical Fluorescence Microscopy for Marine Phytoplankton Count

We propose the use of through-focus scanning optical fluorescence microscopy for marine phytoplankton count. The phytoplankton in a seawater container of 20 ⨯ 20 ⨯ 20 mm is excited by a LED of 435 nm and the fluorescence emission from the phytoplankton over 656 nm is imaged into the image detector (CCD)through an objective lens and a tube lens. The proposed method stacks the sectioned image of the sea water in the container as it optically moves the image plane vertically along the optical axis by moving the tube lens. We built a prototype and it was demonstrated to count the phytoplankton of 1cc (10⨯10⨯10mm)volume with a spatial resolution less than 10 μm in one second.