“You are sitting at your microscope working at high magnification trying to sort out the three-dimensional compartmentalization of your fluorescent probe. You constantly focus up and down bringing different levels of the specimen into sharp focus while letting other levels above and below go out of focus. As this frustrating job continues, you say to yourself “It would be wonderful if I could see the whole specimen in clear focus all at once”. Confocal Laser Scanning Microscopy (CLSM) is the answer. ”

This beautiful passage from C. Robert Bagnell, Jr., Ph.D.’s introduction chapter for CLSM peaked my interest that I’m the one trying to create lab-based microscopy images with unparalleled depth and clarity. Well then, I had the good fortune to work with this incredible machine, which I like to refer to as the “James Webb of The Cell.”

Confocal microscopy technologies have been around for more than 40 years, but the growth and adaptability of the method have been fueled by improvements in optical design, spinning-disk technology, and camera technology.

In contrast to a wide-field microscope, where the entire specimen is illuminated by mercury or xenon and the image is visible to the naked eye, confocal microscopes use a different mechanism for creating images.

Illustrated in the figure below are a series of images that compare selected view-fields in traditional wide-field and laser scanning confocal fluorescence microscopy.