Here a work flow towards an accurate representation of interference colours (Michel-Lévy chart) digitally captured on a polarised light microscope using dry and oil immersion objectives is presented. The work flow includes accurate rendering of interference colours considering the colour temperature of the light source of the microscope and chromatic adaptation to white points of RGB colour spaces as well as the colour correction of the camera using readily available colour targets. The quality of different colour correction profiles was tested independently on an IT8.7/1 target. The best performing profile was using the XYZ cLUT algorithm and it revealed a ΔE of 1.9 (6.4 no profile) at 5× and 1.1 (8.4 no profile) at 100× magnification, respectively. The overall performance of the workflow was tested by comparing rendered interference colours with colour-corrected images of a quartz wedge captured over a retardation range from 80-2500 nm at 5× magnification. Uncorrected images of the quartz wedge in sRGB colour space revealed a mean ΔE of 12.3, which could be reduced to a mean of 4.9 by applying a camera correction profile based on an IT8.7/1 target and the Matrix only algorithm (ΔE < 1.0 signifies colour differences imperceptible by the human eye). ΔE varied significantly over the retardation range of 80-2500 nm of the quartz wedge, but the reasons for this variation is not well understood and the quality of colour correction might be further improved in future by using custom made colour targets specifically designed for the analysis of high-order interference colours.

Coccolith mass is an important parameter for estimating coccolithophore contribution to carbonate sedimentation, organic carbon ballasting and coccolithophore calcification. Single coccolith mass is often estimated based on the k s model, which assumes that length and thickness increase proportionally. To evaluate this assumption, this study compared coccolith length, thickness, and mass of seven Emiliania huxleyi strains and one Gephyrocapsa oceanica strain grown in 25, 34, and 44 salinity artificial seawater. While coccolith length increased with salinity in four E. huxleyi strains, thickness did not increase significantly with salinity in three of these strains. Only G. oceanica showed a consistent increase in length with salinity that was accompanied by an increase in thickness. Coccolith length and thickness was also not correlated in 14 of 24 individual experiments, and in the experiments in which there was a positive relationship r 2 was low (<0.4). Because thickness did not increase with length in E. huxleyi , the increase in mass was less than expected from the k s model, and thus, mass can not be accurately estimated from coccolith length alone.

Different morphotypes of the abundant marine calcifying algal species Emiliania huxleyi are commonly linked to various degrees of E. huxleyi calcification, but few studies have been done to validate this assumption. This study investigated therefore whether E. huxleyi morphotypes can be related to coccolithophore calcification and coccolith mass. Samples from January (high productivity) and September (low productivity) 1997 at an open ocean and a coastal site near the Canary Islands were analysed using a combination of thickness measurements (Circular Polarizer Retardation estimates (CPR) method), Scanning Electron Microscope imaging, and Markov Chain Monte Carlo (MCMC) models. Mean E. huxleyi coccolith mass varied from a maximum of 2.9pg at the open ocean station in January to a minimum of 1.7pg in September at both stations. In contrast, overall calcite produced by E. huxleyi (assuming 23 coccoliths/cell) varied from a maximum of 2.6 μgL-1 at the coastal station in January to a minimum of 0.5 μgL-1 in September at the open ocean site. The relative abundance of "Overcalcified" Type A, Type A, Group B and malformed coccoliths was determined from SEM images. The mean coccolith mass of "Overcalcified" Type A was 2.0pg using the CPR-method, while mean mass of Type A and Group B coccoliths was determined using coccolith length measurements from SEM images and MCMC models relating thickness measurements to morphotype relative abundance. Type A cocccolith mass varied from a 1.6pg to 2.6pg and Group B coccolith mass varied from 1.5pg to 2.0pg. These results demonstrate that the coccolith mass of Type A, "Overcalcified" Type A, and Group B do not differ systematically and there is no systematic relationship between relative abundance of a morphotype and the overall calcite production of E. huxleyi. Therefore, morphotype appearance and relative abundance can not be uniformly used as reliable indicators of E. huxleyi calcification or calcite production.

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Summary This study presents a simple technique for the approximation of retardation, thickness and mass of birefringent particles with a retardation from 8 to 231 nm retardation. Tuning of the imaging system (standard light microscope equipped with a left and a right circular polarizer) to match grey values of polymer retarder films of known retardation with rendered grey values allows for a robust calibration and accurate approximation of retardation. In addition, a technique for accurate particle segmentation using a Canny–Deriche algorithm was used to minimize the bias on mass estimated from different thresholding techniques. The technique was tested using microscopic calcitic plates called coccoliths produced by the marine algal group coccolithophores, and the results compare well with published coccolith mass estimates obtained from volumetric analysis. Lay Description Material with certain optical properties display interference colours when observed in a light microscope under circular polarized light. This study presents a simple technique for measuring the thickness and retardation of small particles within the 8 to 231 nm retardation range based on the grey values of their interference colours. Retardation is a measure of the distance between waves of two mutually perpendicular polarized light waves after passing through material. The technique involves the tuning of a standard light microscope system equipped with a left and a right circular polarizer and a digital camera to match grey values of polymer retarder films with a known retardation with grey values of a digitially rendered Michel‐Lévy chart. A technique for accurate isolation of particles from the image background using a Canny‐Deriche algorithm is also described, which avoids possible biased results from thresholding. The techniques were tested using microscopic calcitic plates called coccoliths produced by the marine algal group coccolithophores, and the results compare well with published estimates obtained from volumetric analysis.