Visualization and quantification of plankton and detritus using digital confocal microscopy

Abstract

The application of digital confocal microscopy to studies of plankton and detritus is described. Plankton cells are fluorescently stained using DAPI and proflavin according to established procedures, while detritus is uniquely stained with DAPI and propidium iodide using a recently published companion protocol. Stacks of digitized images of plankton and detritus are acquired in 3 dimensions (3D) using an integrating color charge-coupled device (CCD) mounted atop a fluorescence microscope. A desktop computer drives a z-axis motorized controller to optically section plankton cells and detritus at very fine intervals (as small as 1 μm or less). Out-of-focus haze associated with each optical slice is removed via a nearest-neighbor algorithm in a process termed deconvolution. Image sets containing these stacks of deconvolved optical slices are subsequently displayed in 3D by volume-rendering software operating aboard a graphics computer. Since the exact x/y/z dimensions of each 3D picture element, or voxel, are known, the volume of plankton or detritus can be calculated A procedure is described whereby the volume of detritus can be converted to units of carbon and nitrogen. This approach, combined with more traditional 2D image analysis of plankton communities, offers the first opportunity to separately quantify the pool sizes of plankton and detritus in aquatic ecosystems.