On Multidimensional Dynamic Fluorescence Imaging and Quantitative Image Analysis in Wide-Field Microscopy Applications to Studies of Astroglial Cells

Abstract: Modern biological research has benefited from a renaissance in light microscopy, brought about by the convergence of developments in fields as diverse as electronics, optics, molecular biology, computer science, and reagent chemistry. Integration of these has transformed microscopy into a highly useful, dynamic research tool for biology and medicine. Now it is possible to generate highly resolved functional maps of molecular events in living systems where new types of specimens can be studied quantitatively and non-invasively, with greatly improved spatial and temporal resolution. By micromanipulation techniques in combination with fluorescence microscopy, it is possible to simultaneously visualize, analyze and perturb cell physiology.

Newly discovered cellular signaling systems in the brain are investigated in a multidisciplinary research program. The astrocytes, the most common glial cells, form an extraneuronal cell network in the brain. The main objectives of the program are to investigate these cell networks and to find out about their roles. The investigations require sophisticated techniques and analysis methods based on, and complementing, conventional fluorescence microscopy. These techniques and methods were developed through medical-engineering teamwork.

In this study, new methodology was developed for studying the characteristics of intra- and intercellular calcium signaling. An automated fluorescence microscopy imaging and image analysis system was developed for this purpose. The image analysis techniques were based on fluorescence wide-field microscopy images combined with algorithms developed for image segmentation, intensity analyses of fluorescence images and motion analysis. The developed system was used for the study of cell state and spatial propagation of calcium waves in cultured astroglial cells. The cell state was determined by analyzing the non-linear transient part of the cell state. In this relation, the amplitude of Ca²⁺ increase in cells and the velocity between cells participating in the Ca²⁺ waves showed exponential characteristics. The influence of different chemical substances induced in the areas of Ca²⁺ wave propagation was determined. The results of the automated system were compared with manual measurements, which confirmed the reliability of the developed automated system as a new tool for further investigation of the calcium wave.

Furthermore, a new methodology was developed for studying and performing quantitative estimation of single-cell volume changes of astrocytes in primary culture. The methodology was carried out by developing a three-dimensional (3D) automated fluorescence microscopy imaging and image analysis system. A new accelerated reconstruction method (the Fast Maximum Likelihood Expectation Maximization method, or FML-EM) and new segmentation methods were developed to estimate single-cell volume changes. However, a 3D reconstructed image comes with an axial ambiguity, which provides a problem for automatic segmentation. We suggested a segmentation method in which the segmentation is defined as an intensity estimation problem. A 3D binary image was created from the gray level reconstructed image. For each 2D binary slice the Maximizer of the Posterior Marginals (MPM) estimates were computed by a modified fast optimal bayesian algorithm. The MPM estimates supplied information for the 2D segmentation, which in turn supplied information for complexity value calculation for each slice. By means of the complexity values, final 3D segmentation was generated.

The segmentation method was applied for volume estimation of astrocytes. The astrocytes exposed to hypoosmotic stress responds with an increase in volume, and those exposed to hyperosmotic stress responded with a volume decrease. The estimated values of the volumes reflected the swelling and shrinking in a robust way. The study demonstrated a large variability in volume changes among cells, both with respect to morphological origin and with respect to capacity. The results of the automated system were compared with those of manual measurements, which comparison confirmed the reliability of the developed automated system as a new tool for further investigation of single-cell volume change. The sensitivity and reproducibility of the technique were evaluated by using spherical fluorescent beads with known volumes.

Finally, quantitative studying of spatio-temporal single-cell volume changes of astrocytes was proposed by a new methodology. The 3D imaging and image analysis system was further developed for this purpose.