A computer‐aided measuring system for the characterization of yeast populations combining 2D‐image analysis, electronic particle counter, and flow cytometry

Abstract

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry

“…The image cytometric measuring system described previously (9) has been applied here to S. cerevisiae cells from both steady-state and synchronous populations. The volume measurements show how the daughter cells in the population largely follow an exponential volume growth pattern (Fig.…”

“…In the present study, we applied a two-dimensional image analysis and contour rotation program developed for separate measurement of the volumes of buds and mother cells (9). By using the volume measurements of cells from a steady-state population and the cycle times of synchronized cells, we have constructed a volume-time diagram which demonstrates the volume growth of daughter cells and consecutive cycles of parent cells, both for the whole cell and for the mother cell parts.…”

Biomass Quantification by Image Analysis

“…4). A size decrease of 35% was observed by Huls et al [29] on Saccharomyces cerevisiae cells. Environmental Scanning Electron Microscopes (ESEM) operate under less stringent conditions: the gas ionization detector tolerates a pressure of about 100 Pa at 277 K, when standard SEM works under about 1 Pa.…”

“…However the preparation of the samples is difficult: dehydration (because of the high-vacuum in the chamber) and metal-plating (to make the sample conductive) are required by most Scanning Electron Microscopes (SEM). The water content of cells being very high, the dehydration and fixation (usually with osmium tetraoxide) should be conducted very carefully to limit the morphological changes [29]. In the best cases, if the general shape is maintained, shrinkage is observed, making size assessment difficult [30][31][32] (Fig.…”

In situ microscopy for on-line determination of biomass

“…Pons used ellipsoids to approximate the shape of yeast cells and to calculate their volume from the semiaxes (Pons et al 1993). Huls described the contour rotation of small cell segments (Huls et al, 1992). In our approach, we refer to the Pons' method.…”