One of the most prevalent types of microscopy is the optical sectioning microscope (including phase, nomarski and confocal microscope), where the researcher can make 4-dimensional recordings (3D + time) of their biological samples. The Laboratory of Optical and Computational Instrumentation (LOCI) has developed a suite of tools to collect, annotate and analyze 4D data from optical sectioning microscopes. A diagram of this system can be seen in Figure.1.

Fig.1 Basic 4D Digital Microscopy Setup

    This is the basic hardware setup that the researcher used to make the movies.
  1. A video camera was mounted on a microscope.
  2. Then the video signal was sent to an in-line image processor for image enhancement.
  3. After that, the images were stored in a computer.

4D Movie Demo

This is a 4D movie that was composed by 3 focal planes - focal plane 2, 4 and 6 (for demonstration purpose we only picked 3 focal planes to use.). Hit play, then try to go up, down, forward and backward to see how the movie changed with the changing depth and time. For instance, you might see nuclei in focal plane 6 but not in focal plane 2 and 4.

Optical Sectioning Microscopy: Various Focal Planes of a 4D Data Set (mouse-over the titles)

Change in Depth Time

This image is an expansion of a 4D movie like the one you saw in "4D Demo". It consists 4 focal planes with 5 time lapses. A 4-dimensional data set consisting of time-lapse images taken at predefined intervals and optical sections. Researchers can specify the number of optical sections they wish to observe for a given time point and then the 4D system changes focus to obtain each optical section or focal plane. Such optical sectioning not only allows researchers to observe events that might be happening at different planes of focus but will also allow them to create a data set that has the biological events they are studying in the best focus. For example, here, the researcher used 4 focal planes with 5 time intervals for every focal plane. The images you see here are all from a single embryo. Accordingly, each row, from left to right, is the same embryo in a different time lapse. Each column, from top to down, is the same embryo in different focal plane. When the recording was done, he/she needed to pick a focal plane that is the most clearest among all the planes. Here, from top to bottom, the 3rd image might be the best choice, i.e, It allows the biological events to be seen most clearly. However, one drawback to committing to one focal plane is that events taking place in other optical sections are no longer seen. This is the reason why in some of the movies you cannot see the early nuclei clearly because they are out of focus due to the fact they are in another optical section.

Image sources: DeVries, J.H., Thomas, C., White, J. (1996). Four-Dimensional Imaging: Computer Visualization of 3D Movements in Living Specimens. Science, 273, 603-607.