Single‐cell tracking with a reversing flow cytometer

Abstract

We have developed an instrument based on a flow cytometer platform that is capable of tracking individual, suspended cells over extended time periods. The instrument repeatedly moves in a capillary the same volume segment of fluid containing tens to hundreds of suspended cells through the focal point of a laser. Individual cells are then tracked based on the timing of when they cross the laser, and cell properties are measured as in a conventional flow cytometer. Because cells are repeatedly measured the single-cell rates of change can be determined. The developed instrumentation was applied to measure the variability of ABC transporter activity in a population of human cancer cells and the temperature dependence of constitutively expressed Gfp in yeast. A wide range of transport rates can be observed in the cancer cell population while the single-cell Gfp fluorescence in yeast shows pronounced oscillations in response to temperature shifts. These observations are not detectable at the population level. Therefore, such measurements are useful for investigating cell function as they reveal how variable properties of single cells change over time. ' 2010 International Society for Advancement of Cytometry Key termssingle cell dynamics; rates of change; drug resistance IN both medicine and biotechnology, understanding how variability of properties arises and changes over time is of fundamental importance. Variability of properties has been indicated in the progression of certain diseases (1) and in explaining the productivity of chemicals or pharmaceuticals with engineered cultures (2,3). This variability has been generally related to genetic differences, to different positions in the cell cycle, to the exposure of a heterogeneous environment, or to stochastic variations due to the low number of molecules in individual cells. While the variability in the single-cell composition can be readily evaluated using conventional flow cytometry, the variability in dynamic properties of cells that reflect the rate at which cells change, are rather difficult to obtain and few examples exist in the literature.To experimentally measure the rate of how properties change, one must measure a property using either an Eulerian or Lagrangian reference frame (4,5). In the Eulerian reference frame one measures the distribution of a property in a cell population at discrete time points. In contrast, in the Lagrangian reference frame one follows individual cells over time and observes how their properties change. The properties of the entire cell population are then obtained as the sum of contributions of the individually tracked cells. The two reference frames are analogous to the fluid mechanic problem of converting Eulerian observations to Lagrangian (4).In the Eulerian reference frame, flow cytometry is primarily used to generate experimental data. This instrument yields a snapshot of the cellular property distribution at different time points and cells are discarded after the measurement. The ability to accurately understand...

Advances in automated real‐time flow cytometry for monitoring of bioreactor processes

“…The distribution of GFP formation was broad for E. coli cells including non‐producer and cells that produced significantly larger amounts than the average cells. Similar results were found in S. cerevisiae cultures [ 1 , 31 , 54 ] as well as that constitutive expression of GFP is highly temperature dependent leading to oscillations [ 153 ]. ART‐FCM was also a rapid method to test promoter strength, plasmid stability and culture variability [ 30 ] demonstrating that small genetic changes could result in large variations in product formation.…”

“…The current state of single-cell analysis involving microfluidics has been reviewed [20,[163][164][165]. In this context novel highly efficient microfluidics based FCs [153,166,167], microfluidic FIA systems [168] and microfluidic fluorescence-activated droplet sorter [117,169] are emerging. As comparability with conventional FCM studies could be shown [165,170], these devices bear great future potential as ART-FCM on a chip.…”

“…Some studies solely applied SYBR Green to discriminate bacteria from background [ 46 , 49 ] or dual staining with PI and FITC labeled Annexin‐V to investigate apoptosis and necrosis in CHO cell cultures [ 38 ]. With FITC alone cell size changes triggered by ethanol and temperature dependence of constitutively expressed GFP in yeast fed‐batch processes could be evaluated [ 55 , 153 ].…”

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

“…One of the main drawbacks of actual on-line FC is its inability to follow the history of given cells inside the population (which makes it difficult to experimentally validate the two hypotheses behind heterogeneity in bioprocesses evoked in Section 2). Some attempts have been made for the adaptation of a conventional FC for the following of certain cells by recirculation of the sheath fluid [81], but this kind of system is difficult to interface with a bioreactor. At this time, the most efficient way to monitor the dynamics of a single cell is by the use of time-lapse fluorescence [82].…”