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Need for winning combinations of modalities in cytometry of stem cells

CONVENTIONAL flow cytometry provides population-level cross-section of cellular features at the time of measurements. However, cellular properties might change dynamically. Cosette et al. (1), emphasize the importance of understanding the possible dynamics of population-level morphology changes frozen in flow cytometric scatter plots. To elucidate the kinetics of these changes, the authors combined imaging flow cytometry and time-lapse microscopy. Their article neatly clarifies the relationship between population based snapshots of morphology parameters (roundness vs. area) measured by imaging flow cytometry (measurements made in the space domain) and features continuous recording by time lapse microscopy (measurements made in the time domain) on living cells. The article concludes that the shape of human hematopoietic stem cells might be determined dynamically with lifetimes of approximately half an hour. The most stable phenotypes are recognized even on conventional twodimensional scatter plots if they significantly differ from each other. The need to characterize cell shapes by simple flow cytometric parameters roots back to the golden era, when scatter and fluorescence light attributes of cells were resolved first and assigned to cell shape and orientation parameters, of chicken erythrocytes (2). It was recognized that small signal of forward light scatter (FSC) came from flattened oval cells, when they were facing toward the diode FSC detector presented a smaller cross-sectional area and in this orientation they generated a smaller fluorescence signal. Orthogonal position of scatter and fluorescence detectors and direction to laser exciter explains the two extreme signal amplitudes of orthogonally oriented cells. Characteristic dual scatter and fluorescence signals of the oval shaped chicken erythrocytes were extensively used for calibration standards in early flow cytometers. Light scatter signal is a robust particle sensor, however, FSC is not linearly related to the size of particles thus limiting its utility in cell identifications. On the other hand, direct current or low frequency impedance, based on the Coulter principle, is linearly related to the volume of particles and widely utilized in clinical hemocytometers. Signal of radiofrequency impedances measured at different higher frequencies reveal various features of the internal structure of cells (3). In static conditions it also provides information on cell attachment and cell–cell interactions (4). Extracellular ligand triggered cell signaling provokes countless cellular responses. It might drive, for example, proliferation, differentiation, apoptosis, etc., of primary, progenitor or stem cells, as well as other cell lines. These processes require transmembrane ion fluxes, exoand endocytosis, intracellular cytoskeletal, mitochondrial, and endoplasmic reticulum alterations. Phenotype switches arise from cell size adjustments, surfaceor intracellular-membrane modifications, or cytoplasmic rearrangements, finally producing changes in membrane capacitance, cytoplasmic conductivity, or permittivity. All of these events deeply affect the dielectric properties of cells (3,5), which can be measured with multifrequency flow cytometric impedance measurements providing multiparametric data. These label-free signals efficiently complement each other and make feasible deciphering diverse shapes, types or physiologic states of cells. A cell sorter, equipped with both scatter and impedance based signal detection, could classify cells without any specific label and could be potently harnessed in stem cell biology to collect developmental intermediates.