Fluorescence microscopy calcium measurements

Tsien, R. Y., Rink, T. J. and Poenie, M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6 145-157,1985. 

Okano et al. [84] measured changes in cytoplasmic Ca2+ concentrations in platelets adhering to HEMA-STY block copolymer (HSB) surfaces by means of fluorescence microscopy combined with a high performance image processor. Comparative studies were also carried out with the HEMA-STY random (HSR) copolymer of poly HEMA and polystyrene. Their results showed that cytoplasmic free calcium levels in platelets that were in contact with the HEMA-STY... 

The fluorescence properties of this and related indicators change markedly when Ca2+ is bound (Figure 15.19). These compounds can be introduced into cells to allow measurement of the available Ca2+ concentration in real time through the use of fluorescence microscopy. Such methods allow the direct detection of calcium fluxes and diffusion within living cells in response to the activation of specific signal-transduction pathways (Figure 15.20). 

It seems valuable to speculate on the potential future applications of lifetime-based sensing. In our opinion, the dominant advantages of lifetime-based sensing are that the measurements can be independent of the local probe concentration, and the decay time measurements are mostly self-calibrating. The insensitivity of the lifetime to the local probe concentration enables the use of lifetime-based sensing in flow cytometry (iiO), fluorescence microscopy 25,88,111-112, Lakowicz, J.R. et al. Cell Calcium, in press), and clinicsd chemistry 25,65-66,113, Lakowicz, J.R. et al. SPIE Proceedings 1993, Vol.1895, in press)... 

Measurement of intracellular and extracellular, Ca " concentrations (fluorescence microscopy, flow cytometry and fluorescence spectroscopy). Conjugates with dextrans are used to confine the indicator to the cytosol. Major applications to the study of calcium regulation and transport. 

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARE-1 into polyelectrolyte multilayer capsules. The co-precipitation of calcium caibonate, urease, and dextran followed up by multilayer film coating and Ca- extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10 to 10 M. Sensitivity to urea in concentration range of 10 to 10 M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by confocal fluorescent microscopy. 

Maxfield, E. R. Measurement of vacuolar pH and cytoplasmic calcium in living cells using fluorescence microscopy. Methods Enzymol. 1989, 173, 745-771. 

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