Question

10. Describe and compare confocal microscopy and wide-field microscopy in the space below (1 page limit)

Answer 1

Wide-field microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. A widefield microscope is easier to use, less complicated, and cheaper than a confocal microscope. Widefield microscopes enable imaging of biological samples that are small, dim, and/or alive. Widefield microscopy works well in dynamic situations, such as imaging moving specimens. A disadvantage of widefield microscopes is that they do not do well with thick specimen or samples that scatter light significantly. Widefield systems are easier to house and maintain, and fine as long as the sample is thin for example, a single layer or cultured cells.

Similar to the widefield microscope, the confocal microscope uses fluorescence optics. Instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. Confocal microscopy is recommended for thicker samples or ones that really scatter light. The background rejection from confocal techniques is helpful for tissue, thick specimens, or even in a high-content approach. Some confocal platforms can be used effectively on live specimens as well as live-cell imaging. Confocal is the clear best choice in some cases, like with tissue slices. Confocal microscopy provides more resolution in the z-axis, or depth.