Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

Monitoring of Intracellular Enzyme Kinetic Characteristics of Peripheral Mononuclear Cells in Breast Cancer Patients

“…FDA staining has been long used in studying the cellular kinetic parameters, measurement of total population, and subset characteristics, and for the estimation of different cellular physiological states, such as activation and apoptosis (19,(22)(23)(24)(25)(26)(27). It has been shown that cell populations exhibit wide heterogeneity with respect to their ability to use FDA, dependent on their type, cell cycle stage, and the cell surface status (28 -31).…”

Single-Cell Microbiology: Tools, Technologies, and Applications

“…Although multiple light scatter and fluorescence parameters may be measured, cells pass only once through the system. Because of this, flow cytometry is not suited for timeresolved studies of individual cells (65,68,124,208). An exception may be the microfluidic cell sorter described by Fu et al (84), in which the fluid flow may be stopped or reversed, allowing multiple observations of the same cell, but this technology is not yet widely available.…”

“…Because LSC can be used to make multiple measurements of the same cells, this technique is well suited for the observation of cellular properties as a function of time. Examples include monitoring the kinetics of fluorescence staining in living cells (e.g., substrate uptake, enzyme activity, and dynamic changes in intracellular pH) and observing interactions between neighboring cells (65,68,230). Spatial "addressing" of fluorescent events may facilitate the reexamination of archived samples (65).…”

“…However, because LSC may involve exposing the sample to the excitation source for relatively long periods, photobleaching of fluorescent labels could be problematic for some applications, particularly if multiple scans are required. These effects can, in part, be minimized through the use of low-intensity (microwatt versus milliwatt) illumination sources (68).…”

“…68,2004 SINGLE-CELL MICROBIOLOGY 545 tion. These include thresholding, filtering, edge detection, removal of optical artifacts (e.g.…”