Water Permeability Measurement in Living Cells and Complex Tissues

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102675 ◽

1988 ◽

Vol 46 ◽

pp. 118-119

Author(s):

K. Jacobson ◽

A. Ishihara ◽

B. Holifield ◽

F. Zhang

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Extended Period ◽

Dynamic Structure ◽

Living Cells ◽

Membrane Dynamics ◽

Molecular Cell Biology ◽

Image Averaging ◽

Single Living Cells ◽

Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122496 ◽

1992 ◽

Vol 50 (1) ◽

pp. 418-419

Author(s):

D. L. Taylor

Keyword(s):

Living Cells ◽

Cellular Level ◽

Molecular Techniques ◽

Cellular Functions ◽

Macromolecular Assemblies ◽

Cell Functions ◽

Molecular Events ◽

Yield Information ◽

Full Knowledge ◽

Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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