The Journal of NIH Research
November 1994 VOL 8
Authors: Eric Gruenstein and Jesus Luna
Reviewed: Mitch Villereal, Ph.D. University of Chicago
Introductionion
The methodology for imaging cytoplasmic calcium in individual living cells with ratiometric dyes such as fura-2 is now almost a decade old. During this period, many calcium signal transduction pathways have been found to involve a variety of subtle spatial phenomena, such as intra- and intercellular waves, as well as, complex kinetic behaviors including repetitive spikes and oscillations. For many investigators interested in studying these effects, the cost of the image analysis system (in the range of $60,000 or more with the microscope) has kept this equipment beyond their reach.
Fortunately, a number of recent technological advances have made it possible to substantially reduce the cost of calcium imaging systems while still maintaining the full functionality of the earlier systems. Opportunities for improved technology have occurred in virtually every major component of the calcium imaging system. For instance, with the advent of the Pentium chip and PCI bus technology, the power of the host computer has become sufficient to allow expensive, hardwired image analysis boards to be replaced by simply frame grabbers with little or no sacrifice in speed. Fluorescence capabilities can be added to simple inverted microscopes by incorporating special optics directly to the objective. And the recent introduction of the integrating CCD video cameras allows for low cost cameras to be substituted for the much more expensive intensified cameras. As a bonus the integrating cameras are not damaged by exposure to bright light.
At least one company has already taken advantage of these opportunities. Intracellular Imaging Inc. has developed a complete system for dual wavelength ratiometric imaging (including the microscope) and single wavelength systems for use with dyes such as Fluo-3. The data presented below illustrate the use of these new systems.
Results
Figure 1 shows the cytoplasmic calcium response of human fibroblasts to serum stimulation. Cells were serum deprived for 2 days and then loaded with fura-2 AM, as previously described (1). At the start of the experiment, fetal bovine serum (FBS) was added and images were collected for analysis with the InCa++ imaging system from Intracellular Imaging Inc. The montage displays calcium images at 4 time points selected from a total of 24. Panel (A) 13 sec before FBS addition; Panel (B) 12.6 sec after FBS; Panel (C) 34 sec after FBS; Panel (D) 176 sec after FBS.
The experiment of figure 2A is similar to that of figure 1 except that instead of saving the images for analysis at the completion of the experiment, objects of interest (i.e. cells) were identified at the start of the experiment and their calcium values were calculated and displayed continuously. FBS was added at the asterisk and the responses of two cells from a total of 8 cells analyzed are displayed. Depending on the cells understudy, ratiometric calcium measurements can be obtained as rapidly as 2 times per sec.
Figure 2B shows spontaneous cytoplasmic calcium spiking activity in cultured embryonic rat cortical neurons. Cells were loaded with fluo-3 and analyzed with the InCa system for single wavelength fluorescence imaging. Such single wavelength measurements can be made as rapidly as 5 times per sec.
References:
1. Wahl, M., & E. Gruenstien, "Intracellular free Ca++ in the cell cycle in Human fibroblasts: Transition from G0 to G1 and progression into S phase" Molec Biol Cell 4. 293-302 (1993).
