Image analysis makes possible the examination of viable cells that are growing attached to Petri plates, Lab-Tek chambers, or multi-well culture plates. Cells are first labeled with a fluorescent antibody, dye, or probe. This technique is also used for the analysis of tissue sections or cover glass cultures following fixation.

Image analysis differs from flow cytometry in that an image of the cell can be obtained and stored as a computer file whereas with flow cytometry, individual cells are represented by dots and populations of cells by histograms or contour plots.

When investigators bring samples to the ACAS, they are usually interested in either,
 

Single image analysis. In this example, the investigator will usually have cells on a slide or coverslip and will want to know what the fluorescence distribution is. This is rare since the use of a standard fluorescence microscope can often answer this type of question. The advantage of the image analyzer lies in the fact that the fluorescent image can be stored as digitized information for subsequent quantitative analysis.

Confocal analysis. Used to obtain images with more detail than standard fluorescence microscopy. The result will be either a single image of the cell(s) under investigation or, a series of sections (Z slices) through the entire cell. If Z-slicing is being performed, the slices may be viewed as a matrix in which all of the slices are presented simultaneously in sequence, or the slices may be assembled into a 3D reconstruction of the cell.

Kinetics. Cells are labeled with a fluorescent probe, such as fluo-3 or indo-1 to detect the modulation of intracellular Ca2+, and then examined over time for changes in fluorescence intensity indicative of altered intracellular Ca2+ levels. Altered Ca2+ levels may result from the addition of agonists or ligands under investigation. Analysis may be in the form of image scans, line scans or point scans.

In an image scan, the fluorescence of the entire cell (or cells) is captured so that subcellular distribution can be observed.
Line scans analyze fluorescence only along a pre-determined segment (line) of the cell. The advantage of line scans over image scans is that whereas an image scan may require several seconds for each scan, line scans may be acquired at rates of around 10/sec. With point scans, data can be acquired approximately ten times faster than when line scans are utilized; a disadvantage compared to the line or image scan, is that a much smaller area is analyzed (only one point C an area equivalent to one pixel).