It is now possible to measure luminescence of single PER-LUC labeled cells using cooled CCD cameras. With their high quantum efficiency and low noise, the cameras can extract significant measurements of luminance from the handful of numbers of photons emitted by single cells. Analyzing the image series from such experiments can laborious, however. Each of the many cells must be identified and then tracked through the image series, a difficult prospect given that the cells can move from image to image, and — during the dim phase of the circadian day — can completely disappear from view. Cell Cycle facilitates the image analysis.
Movie Playback. Cell Cycle assembles the image stack into a movie that can be stepped through or played back and forth at different speeds using standard video controls. Watching the movie makes is easy to pick out the cells that are suitable for analysis. The program automatically adjusts the brightness of the video during playback to compensate for the gradual decline in luminescence of the cells over time.
Automated Cell Tracking. A cell can then be identified for analysis simply by clicking and dragging over it in the image (green rectangle in the image below). The program will then track the cell from frame to frame for as long as the cell remains visible, following it across the image as it moves. Once the cell dims too much for it to be tracked, the program stops automatically. The user can then step frame-by-frame until the cell reappears. A single click on the cell body will cue the program to pick up where it left off and track automatically again through another cycle. With this semi-automated process, it only takes a few minutes to identify a cell and follow it through a complete image series. The program then calculates and displays the luminance trace (graph at the top right). By comparison, extracting the same information from a single cell using generic image analysis programs (such as NIH image) can take hours, since it requires manually shifting the region-of-interest frame by frame.
Circadian Analysis. Once a number of cells have been tracked, the traces can be viewed singly or superimposed for comparison (lower image). The luminance data can also be exported to files that can be read by the LumiCycle Analysis program. The LumiCycle Analysis program can then extract circadian parameters (period, phase and damping, goodness of fit) using a variety of spectral methods.