Optical arrangements

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The inverted arrangements in A, B, C and D (below) are intended for use with phytoplankton. For motile species, we will use a thin layer of poly-L-lysine to immobilise the cells on the lower coverslip. A shows a thin layer of sample (cells suspended in sea water) between two coverslips. The laser is positioned above the sample and normal to the x, y plane. The spectrometer fibreoptic is positioned at 30° to the beam of the laser. It is anticipated that this arrangement may not provide sufficient space between the coverslips for the plasma to form. If this proves to be the case, we shall use spacers to increase the depth of the sample and relocate the fibreoptic below the sample, at 55° to the x, y plane (the largest angle that can be realised with the microscope lens in place). This arrangement is shown in part B.

 

With increasing sample depth, there will almost certainly be an increase in signal contamination from the sea water that the cells are suspended within, which may prove difficult to correct for. This will be particularly true with estuarine samples, which are likely to contain significant amounts of organic matter. C shows an optical arrangement that will maximise the fraction of laser energy reaching the cell. Here, the laser and fibreoptic are both positioned at 55° to the x, y plane, forming an angle of 70° between the laser and the fibreoptic. Inevitably, this arrangement will result in the poly-L-lysine immediately below the sample being turned into plasma and contaminating the signal. We shall assess the need to correct for the poly-L-lysine signal by sampling within a cell-free region. The optical arrangement in D employs a mirror to steer the beam of the laser, so that it hits the sample at 90° to the x, y plane. As with the arrangements in parts B and C, the fibreoptic is at 55° to the x, y plane. The main disadvantage with this arrangement is that the mirror will have to be moved in and out of the path between the sample and microscope with each LIBS measurement. It may also prove difficult to concentrate the beam (using a short focal length lens) whilst remaining below the damage threshold of the mirror.
E and F show optical arrangements for measurements of microphytobenthos within intact biofilms. Chlorophyll a fluorescence imaging is the only method that can be used to locate and identify cells within intact biofilms (Oxborough et al. 2000; Oxborough 2005).

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