Gauging the Elusive

How do you gauge the elusive? Two years ago, in her research at the Leibniz Institute of Plant Biochemistry, biologist Dr. Katharina Bürstenbinder faced this challenge. Her research involves proteins, which influence the biosynthesis and degradation of the cytoskeleton. Inside the cell, the cytoskeleton crisscrosses the cytoplasm like a net of guy ropes. Its shape determines the size, shape and stability of the cell. A change in the cytoskeleton precedes any lengthwise growth of cells. In their search for the factors pulling the ropes in this intricate interplay of cell growth and the shaping of cells, the scientists in Halle (Germany) analyzed the epidermal leaf cells of Thale cress Arabidopsis thaliana.

In the process, they were able to create mutations, which modify the cytoskeleton. The mutations lead to differently shaped epidermal leaf cells. The wild type cells kept their typical irregular shapes (looking like pieces of a puzzle). By contrast, the mutated cells were elongated and had fewer emarginations. In some but not in all of the mutated plants, the cell shape had clearly changed. While the epidermal leaf cells appeared to be different, the reason for it was not so obvious. Did the cell margins indeed show a different number of emarginations of different size? Could the change in appearance be nothing but a subjective impression?

In general, are cell emarginations clearly defined by size and shape? Katharina Bürstenbinder wondered about the answer. Are there ways to determine the exact cell shape and surface as well as the type, size and number of emarginations? Her literature search yielded few results. „Here is a large gap in the methodology“, Katharina Bürstenbinder summarized. „So far, human discretion played a large role in all methods to gauge the 3D structure of cells. However, to err is human.“

Until now, scientists examined intricate cell shapes by computer-aided microscopy. In this method, the cell margins are traced by hand with a marker. This manual definition of emarginations and plasma membrane protrusions is very tedious. „Dependent on the selected resolution, it is easy to overlook small emarginations“, states Katharina Bürstenbinder. In the final step, an image analysis program compiles all obtained data. Researchers have to perform several hundred of these microscopic examinations to arrive at a statistically relevant result. While the computer aids in the procedure, the analysis is very time consuming. Furthermore, the results lack precision, and the interpretation can be subjective.

Of course, relying on the subjective impression about the nature of an effect is incompatible with scientific principles, which call for measurability and reproducibility. It is hard to compare effects, which cannot be expressed in numbers or simple parameters. To overcome the above described problems, Katharina Bürstenbinder, Birgit Möller and Yvonne Pöschl joined forces and developed the PaCeQuant application for the quantification of epidermal cell sizes and shapes. Birgit Möller and Yvonne Pöschl are informatics experts from the Halle University. Their expertise contributed significantly to the success of the project. For the development of the PaCeQuant program, the scientists defined 27 different parameters for the determination and calculation of cell shapes. Using defined survey points, PaCeQuant automatically recognizes and calculates parameters such as cell circumference, number and size of the emarginations as well as the cell surface with and without emarginations. Furthermore, the program records and analyzes the data of several hundred cells simultaneously.

Quite a bit of excitement was in the air when the scientists put the method to the test: „We used PaCeQuant to measure the cells. Then we asked several outsiders to measure the same cells the traditional way.“ In traditional measurements, the results deviated by more than 40 percent. When PaCeQuant recorded 20 emarginations, one human experimenter found only 12 emarginations while others found up to 28. Katharina Bürstenbinder is aware of this fact: „These differences in reported data do not only depend on the person who analyzes the cell but also on this person’s level of alertness on a particular day.“
PaCeQuant has many potential applications. From now on, analyzing the shapes of various mutant cells will be an automated process. Mutant cells can be distinguished from each other and also from wild type cells. What is more, the program also recognizes age-related cell shape differences. The program can report data by cell size according to preset parameters for small, medium and large-sized cells. This makes it possible to analyze the number and distribution of cells in different phases of their development. Plant physiologists are now able to distinguish and define the growth stages of plants in even greater detail. Moreover, it will be possible to adapt the program to measuring other cell types in other organisms. Using PaCeQuant for the automated recognition of abnormal cells in cancer diagnostics is another conceivable application.

For the time being, Katharina Bürstenbinder would like to see the program used by as many plant physiologists as possible: „The PaCeQuant image analysis would create a uniform standard for the measurements done for the same cell type by various groups of scientists. This would make it easier for the various groups to compare and discuss their data.“ The first interested parties are already eager to use the program. They already optimized the PaCeQuant program to adjust it to other cell types. The benefits of the open source program are already apparent: Scientists everywhere in the world are able to adapt the program dynamically to their particular projects and requirements.

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