Software tools for 4D live cell microscopy

Abstract

The Integrated Microscopy Resource has developed a trio of Macintosh-based software tools which allow the user to collect digital 4D microscopy (3D over time) of live cells. To demonstrate the program, high-resolution Nomarski DIC images were collected from an inverted microscope using a video camera. The output of the camera can be averaged and contrast-enhanced before the resulting signal is digitized using a framegrabber card installed in a Macintosh computer (Figure 1). Collection, subsequent processing, and analysis of the resulting data sets are handled by the following three software tools:4D Acquisition Software - This IMR-designed software system is based on the popular shareware image processing program “NIH-Image” (author Wayne Rasband, NIH) and controls the actual image acquisition through a series of graphic user interfaces. A framegrabber card, a stage drive motor, and an illumination shutter are manipulated to allow the imaging of either a single focal-plane of the specimen over time, or the acquisition of 3-dimensional volumes at each timepoint. After selecting the top and bottom of the sample, the user can specify the number of focal planes to acquire, the increment for the focus motor between focal planes, the number of timepoints to collect, and a file format in which to save the images. The software will automatically move through the sample from the top to the bottom gathering images of each focal plane as it goes. The result is a stack of images representing the 3D structure of the sample. The software will wait a defined amount of time and the process will be repeated for as many timepoints as the user has designated.