Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
There are many advantages to using a confocal rather than a traditional widefield microscope. The abilities to image optical sections in a series through a specimen, and to eliminate light signals that are out of the plane of focus, have made confocal imaging a popular choice. But potential confocal buyers will find more varieties of confocal microscopes than ever before—how do you begin to choose? One of the most basic yet important factors is the type of specimen you want to study (such as fixed or live cells) and the type of measurements you want to make (for example, static or dynamic cellular processes).
Confocal imaging with fixed cells
For imaging cells or tissues that have been fixed and stained, a laser scanning confocal microscope (LSCM) is the most common choice. This is mainly because imaging fixed cells, which lack the rapid biological events studied in living cells, benefits more from the typically higher spatial resolution of LSCMs. The acquisition rates, which are limited by the movements of the LSCM’s galvanometer mirrors, are approximately 1 frame/second. To take images, the LSCM’s laser makes optical sections by scanning through the specimen. Longer exposure times may mean the danger of some photobleaching of the signal by the laser light. However, with fixed cells, time is not as essential, and one can usually acquire multiple images of a field and average them together.
Confocal imaging with live cells
Imaging live cells entails a certain degree of extra care to guard them from harmful effects of the unfriendly microscopy environment. Keeping your cells alive and healthy is paramount and necessitates consideration of a heating element and perfusion system for their time on the stage, especially for time-lapse studies. You might even need a different confocal microscope. The rapid chemical and biological events that control live cells’ cellular physiology (which, not coincidentally, are also the events most people want to study) are simply too fast to investigate using conventional LSCMs. In addition, the longer light exposures used by LSCMs can cause toxic photo damage to cells.
For confocal imaging of live cells, there are two general options: a modified LSCM or another type of CM altogether. Modified LSCMs come in several varieties—for example, a type that uses resonant scanning mirrors instead of the slower galvanometers can acquire images at about 30 frames/second. Alternatives to LSCMs include spinning disc confocal microscopes (SDCMs), whose main advantage is that they acquire images much faster (though, inevitably, at a cost of some spatial resolution compared to LSCMs). For example, image acquisition rates of an SDCM—also known as a field, array or line scanner—can approach 2,000 frames/second (although often other factors lower this rate somewhat).
If you need to watch out for photobleaching or have weaker fluorescent signals, an SDCM might be best for you. It uses two disks containing many arrays of pinholes that spin or rotate and disperse excitation light. Instead of scanning the specimen using a laser line, like the LSCM, the spinning disk variety collects many points at once. An SDCM typically collects approximately 100 pixels simultaneously, compared to an LSCM’s one pixel at a time. This ability to image the whole field at once results in lower light exposure, and less photobleaching and phototoxicity, with SDCMs.
Automated confocal imaging systems
Some people like to tinker around with parts of microscopes, some need the opportunity to make custom adjustments to their instruments and others want nothing to do with the inner workings of the microscope. Those in the latter camp might consider an automated confocal microscope. One example is Olympus’ FluoView® FV10i, a four-laser confocal microscope that is completely automated, compact enough to fit on a benchtop and doesn’t require its own room. This is because of its construction—a self-contained box that essentially acts as its own tiny darkroom. Systems like this are a great choice for researchers who expect to do a lot of routine, data-chugging imaging that is important but doesn’t use the full expanse of a traditional confocal system’s capabilities or flexibility.
Another example of an automated confocal system is Nikon’s A1, which uses galvanometers commonly used by most laser scanning confocal microscopes. This system offers higher resolution than its cousin, the A1R, which has a hybrid scanner with faster acquisition rates (about 30 frames/second) but lower resolution than the A1. The A1R also allows simultaneous photoactivation and imaging for studying dynamic interactions, such as photoactivatable molecules in intracellular signaling pathways.
Learning more about these general types of confocal microscopes can help you find the right instrument for your research. And even if you don’t want a fully automated system, your lab can probably benefit from the many user-friendly features built into most systems today, whichever model you choose.
The image at the top of the page is from Olympus FV10i.