Authors: Michael Higdon, Intracellular Imaging, Inc. and Eric Gruenstein, University of Cincinnati College of Medicine

Reviewed: P. Darwin Bell, Ph.D., The University of Alabama at Birmingham

So, your research project has finally reachedthe point where you're starting to think seriously aboutwanting to measure intracellular calcium. And now you're asking yourself questions like: How expensive is this going to be? How hard is it going to be learn to use the technology? And which is preferable, imaging or photometry. The answer to the first question is that complete ratiometric imaging systems including fluorescence microscope, low light level video camera, filter changer and image processing computer have recently become available for about $30,000, while complete micro-photometer systems with single wavelength rather than ratiometric capabilities can be had for under $15,000. Concerning the second question, application software for most of these systems is by now available under WindowsTM which helps considerably with the learning curve. But for real ease of use, look for systems that do the things you need without lots of bells and whistles that you will rarely or never call for. The third question, imaging or photometry, requires a little more discussion.

Digital Fluorescence Imaging

    Chief among the advantages of this technique is spatial resolution. It is required for studies involving features such as sub-cellular gradients, intercellular waves, or populations of cells with heterogeneous responses. An example is shown in figure 1 where quiescent human fibroblasts were stimulated to reenter the cell cycle by addition of serum. Cytoplasmic calcium was monitored by fluorescence ratiometry with the dye fura-2. In panel A dual wavelength ratiometric analysis reveals subcellular gradients and waves of cytoplasmic calcium. Panel B reveals the differing calcium kinetics of three cells in a single microscopic field. Imaging permits the separate analysis of each cell. Note the oscillation in one cell and the delayed response in another. Panel C combines the data of all three cells as would be the case for a photometric analysis.

Photometry

    Although photometry provides no spatial resolution beyond selection of the field to be observed, it is superior to imaging in both temporal resolution and sensitivity. This is particularly important for studies of very brief calcium transients such as those of nerve, studies in which the objects may be moving such as contracting muscle, studies in which cells load poorly with dye, or studies on cells particularly sensitive to photodamage. An example of micro-photometry is seen in figure 2 where the kinetics of a cluster of synoptically connected neurons undergoing highly synchronized calcium oscillations is shown. With the calcium sensitive dye fluo-3, photometric measurements can be obtained as rapidly as 200 times per second using only a small fraction of the light intensity required for imaging experiments. However, note also that unsynchronized oscillations would probably not have been detected by photometric analysis.

    In summary, digital fluorescence imaging and micro-photometry are complementary techniques with distinct advantages and disadvantages. If the responses you want to measure last for a few seconds or more and occur in cells that are not unusually sensitive to photodamage, video microscopy with digital image analysis will allow you to detect gradients, waves, and population heterogeneities. On the other hand, if the responses you are interested in are complete within a second or less or require especially low levels of fluorescence, micro-photometry may be the method of choice.

Figure 1a.

Figure 1b.

Figure 1c.

Figure 1. Image analysis showing elevation of cytoplasmic calcium following serum stimulation of human connective-tissue cells using the InCa++ Image Analysis System from Intracellular Imaging, Inc. See text for description.

Figure 2. [Ca++] kinetics in a cluster of spontaneously oscillating neurons loaded with Fluo-3. Data were acquired using the InCaPhot photometry module from Intracellular Imaging, Inc.