Epifluorescence microscopy

This method is used to determine microbial community size and trophic structure for understanding marine food webs. We will use it with our ongoing weekly sampling of Boothbay Harbor, where we observe a strong phytoplankton seasonal cycle, alternating between large diatoms and dinoflagellates, to small picoalgae and flagellates. The priority image analysis problems here are:
1) classification of cells to about 10 common types
2) accurate discrimination of cells from detrital particles
3) separation of overlapping cells and particles

Color epifluorescence images of nanoplankton from the Gulf of Maine, shown here in grayscale. A) the blue-excited image shows all particles labeled with proflavine (yellow-green) and the autofluorescence of plant pigments (red and orange). B) the UV image shows the location of DAPIlabeled DNA in cell nucleii as bright areas in the cells. An expert has identified cells of interest by drawing boxes around them. The numerous, small bacteria cells were not included in this analysis. Note that the larger detrital particle in the lower left of A does not contain DNA. Note problem of overlapping cells. These two images show 4 of the 5 image planes used (green excitation is not shown). Scale bar is 10 m.