Chapter 5: Imaging in depth: controversies and opportunities.

One enduring challenge of biological imaging is achieving depth of penetration-into cells, tissues, and animals. How deeply can we probe and with what resolution and efficacy? These are critical issues as microscopists seek to push ever deeper, while resolving structural details and observing specific molecular events. In this guide to depth-appropriate modalities, standard optical platforms such as confocal and two-photon microscopes are considered along with complementary imaging modalities that range in depth of penetration. After an introduction to basic techniques, the trade-offs and limitations that distinguish competing technologies are considered, with emphasis on the visualization of subcellular structures and dynamic events. Not surprisingly, there are differences of opinion regarding imaging technologies, as highlighted in a section on point-scanning and Nipkow-disk style confocal microscopes. Confocal microscopy is then contrasted with deconvolution and multi-photon imaging modalities. It is also important to consider the detectors used by current instruments (such as PMTs and CCD cameras). Ultimately specimen properties, in conjunction with instrumentation, determine the depth at which subcellular operations and larger-scale biological processes can be visualized. Relative advantages are mentioned in the context of experiment planning and instrument-purchase decisions. Given the rate at which new optical techniques are being invented, this report should be viewed as a snapshot of current capabilities, with the goal of providing a framework for thinking about new developments.