Multidimensional single-cell analysis based on fluorescence microscopy and automated image analysis

Abstract

A single-cell analytical technology was developed for evaluating fast-growing cultures of green algae. The main part of the single-cell analysis is an epifluorescence microscopy-based cytometric approach combined with an automated image analysis algorithm and a single-threshold discrimination procedure. The reliability of the technique in terms of object recognition, evaluating particle size, and determining chlorophyll was successfully proven via reference analyses. The microscopy technique was used to determine the size of single cells, the amount of chlorophyll, and the density of chlorophyll in a model algal culture (Acutodesmus o.). The algal cells showed unexpected heterogeneity in all single-cell parameters, and exhibited a high correlation between cell size and amount of chlorophyll but a very low correlation between cell size and chlorophyll density. For a given cell size, the cell-to-cell heterogeneity of the relative chlorophyll density showed a spread of 0.02-0.08. This points to large variations in the architecture and the physiological state of the photosynthetic apparatus in the cells. This complex situation should be considered in future systems biology approaches focusing on the relationships between biomass accumulation, photosynthetic activity, and central carbon metabolism. Graphical abstract Analysis of cell-to-cell heterogeneity obtained from microscopic images.

Analysis of population structures of the microalga Acutodesmus obliquus during lipid production using multi-dimensional single-cell analysis

“…Cell size determination was conducted after image processing with an up-stream contrast enhancement method. This procedure artificially increased the light intensity that is scattered inside the non-fluorescent cell volume and thus, enables a proper determination of the cell size 19 , 23 . Following contrast enhancement, the discrimination between objects and background was done with a single threshold procedure combined with an optimization step to define the threshold 28 .…”

“…A linear correlation with a R 2 of 0.963 was observed. Additionally, reliability of the object recognition capabilities of the algorithm was recently proven with different particle size standards and with cells from A. obliquus 19 . Some physical phenomena can disturb the optical analysis of TAG and chlorophyll in algal cells.…”

“…Raw images were processed using a custom-designed routine in MATLAB (The MathWorks, Natick, USA). Object recognition steps and technical details have been described elsewhere 19 . The established routine was extended to include cell size determination, chlorophyll amount and content and lipid amount and content as primary signals followed by distribution characterization and correlation analysis steps.…”

“…A deep insight of the technical advantages and disadvantages of FC is given by Shapiro (2003) 17 . In addition to FC, microscopy-based single-cell analysis and automated object recognition became an emerging tool for SCA 19 – 22 . The advantages of this so-called microscopic cytometry in comparison to FC, are direct access to cellular size and morphological properties that are intrinsically embedded within the microscopic images and the access on spatial information enabling subcellular analysis.…”

Probing the changes of carotenoids in Microcystis flosaquae under environmental perturbations by two‐dimensional Raman correlation spectroscopy

“…Traditionally, the algal intracellular characteristics, including the components, metabolites, and evolution of the composition upon environmental perturbations, are measured with established analytical methods such as fluorescence microscopy, ultraviolet absorption spectroscopy, high performance liquid chromatography and mass spectrometry . Additionally, the algal physiological responses to different environments are analyzed by taking a single cell or populations into consideration .…”