How can you choose the right confocal microscope for you? Is there any specific method?
Confocal microscopy has more advantages than widefield microscopy. Confocal microscopy can do continuous optical sectioning of samples and exclude signal interference from non-focal planes. For this reason, the application of confocal microscopy is indeed more common. However, there are more and more confocal microscopes on the market. How to choose?
Confocal microscopy has more advantages than widefield microscopy. Confocal microscopy can make continuous optical sections of samples and exclude signal interference from non-focal planes. For this reason, confocal microscopy is indeed more common. However, there are more and more confocal microscopes on the market. How to choose? When choosing a confocal microscope, there are two basic and important considerations: the type of sample you want to study (eg, fixed or live cells), and the type of assay you want to perform (eg, static or dynamic cellular processes).
Confocal imaging of fixed cells
To image fixed and stained cells or tissues, we generally choose laser scanning confocal (LSCM). This is mainly because fixed dead cells lack rapid biological events in live cells and can better benefit from the higher spatial resolution of LSCM. LSCM imaging is to optically slice the sample by laser, and the scanning speed (depending on the speed of the scanning array mirror) is generally 1fps. Longer exposure times mean a greater risk of photobleaching, but for fixed dead cells time is not critical and we can average it out by taking multiple pictures.
Confocal imaging of live cells
Imaging live cells requires additional protection from unfriendly environments. When imaging, our first consideration is to maintain cell viability and health. Constant temperature heating elements and perfusion systems are essential, especially for time-lapse studies. The fast biochemical events that control the physiological state of living cells are what most people want to study, but these events are too fast for conventional LSCM. In addition, prolonged exposure to LSCM also induced toxic light damage to cells. Therefore, imaging live cells requires special confocal microscopes.
There are generally two options for confocal imaging of living cells: fast scanning laser scanning confocals and spinning disk confocals. Fast scanning confocal microscopy replaces slower galvanometersmirrors with faster resonant scanning mirrors, enabling scanning speeds of up to 30fps; while spinning disk confocals (SDCMs) have the advantage of faster scanning (although inevitably at a loss Spatial resolution), its speed can theoretically reach 2000fps (in practice it is often limited by other factors).
If you are concerned about photobleaching or very weak fluorescence signal, then SDCM may be more suitable. SDCM uses two rotating discs with arrayed microwells to disperse the excitation light. Unlike LSCM, which scans the sample spot, SDCM acquires multiple spots at a time (about 100 pixel spots), so the speed is greatly increased, and the photobleaching of SDCM and less phototoxicity. However, you can also use fast scanning confocals to increase scanning speed, combined with highly sensitive detectors, and reduce excitation light energy to reduce phototoxicity, which can maintain the rich functions of traditional confocals.
Automated confocal imaging system
Some researchers like to find ways to modify the microscope to suit their individual needs. And some researchers aren't even interested in how microscopes actually work. For the latter, an automated confocal microscope may be more to their liking. For example, the Olympus FluoViewFV10i is such a fully automatic desktop 4-laser confocal microscope. Its compact integrated structure requires almost no installation space. This type of instrument is ideal for researchers who need to do a lot of image data acquisition every day, but of course they do not fully reflect the functionality and flexibility of traditional confocals. In addition, the NikonA1 is also a fully automatic confocal system. Like most conventional confocal scanning microscopes, the NikonA1 uses a galvanometer scan head, which has a higher resolution than the A1R with a hybrid scan head, but a slightly slower speed (A1R ~30fps)
