Measurement of protein concentration in bacteria and small organelles under a light transmission microscope.

Protein concentration (PC) is an essential characteristic of cells and organelles; it determines the extent of macromolecular crowding effects and serves as a sensitive indicator of cellular health. A simple and direct way to quantify PC is provided by brightfield-based transport-of-intensity equation (TIE) imaging combined with volume measurements. However, since TIE is based on geometric optics, its applicability to micrometer-sized particles is not clear. Here, we show that TIE can be used on particles with sizes comparable to the wavelength. At the same time, we introduce a new ImageJ plugin that allows TIE image processing without resorting to advanced mathematical programs. To convert TIE data to PC, knowledge of particle volumes is essential. The volumes of bacteria or other isolated particles can be measured by displacement of an external absorbing dye ("transmission-through-dye" or TTD microscopy), and for spherical intracellular particles, volumes can be estimated from their diameters. We illustrate the use of TIE on Escherichia coli, mammalian nucleoli, and nucleolar fibrillar centers. The method is easy to use and achieves high spatial resolution.

Model MA ，Guo R ，Fasina K ，Jin R ，Clements RJ ，Leff LG ... -  《-》

Macromolecular crowding: chemistry and physics meet biology (Ascona, Switzerland, 10-14 June 2012).

Foffi G ，Pastore A ，Piazza F ，Temussi PA ... -  《-》

Comparison of cell volume measurements by fluorescence and absorption exclusion microscopy.

There are two light microscopic methods for cell volume measurement based on volume exclusion. One method, sometimes referred to as FLEX, utilises negative staining by an external fluorescent dye, and cell volume is found from attenuation of fluorescence under a wide-field microscope. The other method (TTD) is based on exclusion of an external absorbing dye, resulting in an increased intensity of transmission image. In this work, we compared these two methods. TTD measurements were consistent, reproducible and identical to those obtained by confocal scanning. In our hands, FLEX based on either sodium fluorescein of fluorescent dextran, usually resulted in underestimation of cell volume, which were insignificant in shallow chambers but became more severe with increased chamber depth. We have not been able to exactly pinpoint the source of the problem; it may have been undetected accumulation of dye in the cells or, more likely, some unappreciated aspects of image formation under epi-illumination. We also discuss applicability of both methods to in-flow volume measurements. LAY DESCRIPTION: Cell volume is a parameter important for many cell biological and physiological applications, and many different methods have been proposed for its measurement. Two light microscopic methods based on volume exclusion deserve special attention due to their speed and simplicity. In one of them (transmission-through-dye, or TTD), cells are placed in a shallow chamber, and a strongly absorbing external dye is added to the cell-containing medium. The sample is imaged in transmission at a wavelength of maximum dye absorption. Because cells with intact membranes exclude the dye, they appear brighter on a transmission image, and their contrast quantitatively reflects cell thickness. By summation of thickness values over the cell area, cell volume can be obtained. The other method sometimes referred to as FLEX utilises exclusion of a fluorescent dye. Cells appear darker than the background under wide-field fluorescence observation in accordance with their thicknesses, and cell volume is computed by thickness summation over the area, like in TTD. In this work, we compared the accuracy of TTD and FLEX for volume measurements. TTD and confocal scanning produced virtually identical results, which suggests that TTD data are accurate. On the other hand, cell volumes measured by FLEX were consistently smaller than by TTD. The discrepancy always increased with the depth of the chamber, although the exact relationship varied between experiments. By contrast, TTD results were insensitive to chamber depth. Thus, it appears that FLEX underestimates cell volume. The reason for that is not entirely clear. Accumulation of the fluorescent dye inside the cell could be a possibility, although we found no evidence for that. Most likely, the reason lies with some unappreciated aspects of wide-field fluorescence image formation, which has not been well-characterised for the type of negative staining used in FLEX. In our opinion, TTD is the method of choice, at least for stationary cells. On the other hand, due to linear dependence of intensity on volume, FLEX might offer advantages for high-throughput flow volume imaging, although realisation of such an experiment has yet to be worked out.

Model M 《-》

Calibrated brightfield-based imaging for measuring intracellular protein concentration.

Intracellular protein concentration is an essential cell characteristic, which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/L). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (μm3 ) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Because interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle, we measured this parameter in three cell lines: HeLa, MDCK and DU145. Our data indicate that protein concentration remains relatively stable in these cells. © 2017 International Society for Advancement of Cytometry.

Mudrak NJ ，Rana PS ，Model MA 《-》

Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect.

Coquel AS ，Jacob JP ，Primet M ，Demarez A ，Dimiccoli M ，Julou T ，Moisan L ，Lindner AB ，Berry H ... -  《-》