2. Image Analysis

3. Flow & Image

4. The present

5. The future

This presentation will examine the traditional roles that both flow and image cytometry have played traditionally and more recently, the links that have become evident. Both technologies are technically demanding, scientifically complex, have unavoidable deficiencies, but have become vitally important in the biological sciences.

We will examine the basis for the success of flow cytometry, both in the earlier days and in more recent times. Looking back on 25 years of history gives us an interesting perspective on this fascinating technology. The dramatic increase in usage due to AIDS related disease provided a major boost at a time when it had perhaps become somewhat routine. Concomitant with a dramatic increase in production and sales of clinical instruments there was also a steady increase in flow cytometry related publications in the literature.

However, other uses for flow cytometry were also being discovered building up a very large data base of capabilities. It is perhaps these capabilities that will carry the technology into the 21st century as a tool easy to use, and as useful as a regular lab centrifuge. Perhaps the growth of image cytometry will be one of the driving forces that will maintain flow cytometry as an important technology in the future.

Image analysis predated flow cytometry as a science, and was in fact a driving force that led to much of the subsequent developments in flow cytometry. The most difficult problem to be faced by "image" scientists was clearly the lack of computational power. While traditional image analysis has been available for many years, it has only been readily available as an economically viable alternative to the majority of scientist for about 5 years. The recent growth of confocal microscopes and less expensive comprehensive image systems, is probably far surpassing the growth of flow cytometry in the 70s and 80s. Powerful computers, now available at very low cost, far exceed the computational capability of supercomputers designed just 10 years ago. The incredible increase in technology and reduction in cost of video systems are two factors which have reduced image analysis to a standard laboratory technology.

It is interesting to observe the growth of image technologies in traditional flow cytometry laboratories. Many flow cytometrists have for years been denied easy observation of material run on their cytometers. Of course, it is easy to place specimens under a microscope, something done all to infrequently! However, there are many situations where both technologies, when available, complement each other tremendously.

As a simple example, the study of endothelial cell function by flow cytometry requires either breaking up of cell monolayers or removal of cells from tissue. While cell cultures are not ideal for the study endothelial (or any other cell ) function, the technique at least allows evaluation of the cells in a situation which mimics to some extent, the natural host. To evaluate surface markers, intracellular function such as calcium fluxes, or reactive oxygen or reactive nitrogen species by flow cytometry, the spatial relationships between cells and their surfaces must be destroyed to provide single cell suspensions. A tremendous amount of population information can be gleaned from such an experiment within a couple of hours. Image analysis on the other hand, allows an extensive evaluation of fewer cells, but much more information is gleaned regarding cellular relationships, morphological information and the like.

Further, continuous analysis of the exact same cells can be accomplished - something impossible in flow at the present time. Single cell recordings of changes in physiological phenomenon can be performed with ease; photobleaching and recovery of fluorescence, as well as UV based release of caged compounds can also be achieved, while monitoring a single or even several cells simultaneously. While achieving many of these studies, using confocal technologies, it is also possible to locate with some specificity intracellular organelle location for the phenomenon under study.

From a practical point of view, it is clear that the two technologies are complementary and the two capabilities provide wonderful synergy when combined. Since the biology, the fluorescence chemistry and the scientists can be a common thread in these equations, it appears that there is a very good chance of success in mixing the two technologies in the working flow environment.