Fluorescence Ca2 Imaging

2.1.1. Background

Fluorescence is the result of a three-stage process involving both the absorption and release of energy (Fig. 1A). A photon of energy (hvex) supplied by an external light source (lamp or laser typically) is absorbed by the fluorophore. The activated fluorophore remains in this excited state (S,') for a finite time (1-I0xl0"9 sec), during which it undergoes conformational changes until it reaches a more relaxed state (S,). A photon of energy (hvem) is emitted when the fluorophore returns to its ground state (S0); the wavelength of the emitted light ("fluorescence") is generally longer due to energy dissipation during the transition from the S,' to S, states. Typically many fluorophore molecules are excited simultaneously to variables,, states, then return to slightly different levels of S,, resulting in a wide emission spectrum. Fluorescent 2+

indicators for Ca are typically chelators that change or shift their fluorescence spectrum when bound to their ligand, Ca2+.

Figure 1. Fluorescence properties and fluorophore loading. A: Jablonski diagram depicting the excitation of fluorescent indicators by energy absorption (hvex), the generation of the excited and relaxed singlet states (S,' and S,, respectively), and the subsequent emission of fluorescence coupled to the return of the fluorophore to its ground state (hvcx). B: Schematic representation of active (left panel) and passive (right panel) loading techniques. In active loading, the indicator is directly introduced into single cells, as shown by pipette injection in this figure. Use of acetoxymethyl ester indicators (shown here) is described in the text. Both loading techniques result in a significant number of fluorophore molecules binding to free Ca2+ within the cytoplasm (bottom panel), as well as a number of unbound fluorophores (which are washed from the cell prior to experimentation and free Ca2+ ions (unbound by fluorophores) which will not fluoresce.

Figure 1. Fluorescence properties and fluorophore loading. A: Jablonski diagram depicting the excitation of fluorescent indicators by energy absorption (hvex), the generation of the excited and relaxed singlet states (S,' and S,, respectively), and the subsequent emission of fluorescence coupled to the return of the fluorophore to its ground state (hvcx). B: Schematic representation of active (left panel) and passive (right panel) loading techniques. In active loading, the indicator is directly introduced into single cells, as shown by pipette injection in this figure. Use of acetoxymethyl ester indicators (shown here) is described in the text. Both loading techniques result in a significant number of fluorophore molecules binding to free Ca2+ within the cytoplasm (bottom panel), as well as a number of unbound fluorophores (which are washed from the cell prior to experimentation and free Ca2+ ions (unbound by fluorophores) which will not fluoresce.

Fluorescence output of the indicator is of key importance in their use. Fluorescent dyes are inherently sensitive to decay due, in part, to repeated exposure to excitatory wavelengths ("photobleaching"). Photobleaching can be avoided by using low-light detection devices such as CCD cameras or photomultiplier tubes (PMT), high numerical aperture objectives that capture a larger area of emitted light such that the excitation intensity can be reduced. Simply increasing the number of fluorophores available for detection (see loading strategies below) can significantly enhance emission, although this approach may increase the rate of self-quenching (quenching of one fluorophore by another) and/or fluorescence resonance energy transfer (where emission of one fluorophores is coupled to the excitation of another). The indicator should be carefully chosen for its absorption and emission properties (molar extinction coefficient for absorption and quantum yield for fluorescence, respectively) to maximize the efficiency of the detection tools.

2.1.2. Loading Techniques

The cell loading of intracellular dyes is generally divided into two groups. Bulk loading procedures by chemical, mechanical and electrical means apply to large populations of cells and include acetoxymethyl ester form loading, detergent- or ATP-induced membrane permeabilization, liposome delivery, electroporation, or hypoosmotic shock. Single-cell loading procedures involve the injection of dyes into one cell at a time (5, 6) (Fig. 1B, left). The use of non-invasive AM ester form of the dyes (Fig. 1B, right) is the most popular method for loading fluorescent indicators. In this process, the charged moieties of a fluorescent indicator are bound to an acetoxymethyl (AM) group via an ester bond, rendering the indicator non-polar allowing it to permeate the cell membrane. In the AM form, fluorescent dyes are insensitive to ions. Incubation of the dye (dissolved in anhydrous DMSO, then added to media) at room or body temperature for 15-60 minutes allows for membrane permeation. Once inside the cells, AM esters are cleaved by intracellular esterases, making the dye sensitive to its target molecules, and trapping it within the cell.

In the case of patch-clamp electrophysiology, both bulk and single-cell techniques have been successfully used. Because some dyes are not available in membrane-permeable forms, electrophysiologists can also incorporate fluorescent dyes into the pipette solutions. Upon obtaining whole-cell access, fluorophores dialyze into the cytosolic space and bind their targets (Fig. 1B, left). The use of non-permeable dyes avoids problems associated with AM dyes, such as compartmentalization into intracellular organelles, incomplete AM ester hydrolysis, and extrusion by organic ion transporters.

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