Disk-Scanning Confocal Microscopy

Abstract

Rapid biological imaging of faint fluorophores in living cells — especially in four dimensions [three dimensions + time] — imposes different instrumentation challenges from slowly acquiring a single high-resolution confocal snapshot of fixed tissue. High acquisition speeds with acceptable contrast and minimal photobleaching suddenly become essential, all without losing the instantaneous optical sectioning that a confocal microscope affords. Of particular interest here, disk-scanning confocal microscopes are proving to be a powerful tool in rapid imaging of live cells in space and time. While the principle is relatively old, new instrument developments, both in optics and in novel ultra-sensitive chargecoupled device (CCD) cameras are greatly expanding the versatility and scope of this approach. However, knowing which system is best for a given question and understanding the inherent strengths and weaknesses is not easy, especially as many of the choices involve complex trade-offs between resolution, speed, sensitivity, 1 and signal-to-noise ratio (S/N). The goal of this chapter is to provide both a theoretical and a practical guide, with more emphasis on the latter especially when theoretical considerations are covered elsewhere (Chapters 22, 23, and 34, this volume). Here the relative merits, strengths, and weaknesses of using a disk-scanning confocal microscope for biological imaging will be examined and sample applications shown.