Live-cell imaging is a powerful technique that allows you to image dynamic cell processes, such as protein trafficking, signal transduction, endocytosis, and exocytosis, etc. However, to perform your own live-cell imaging you need the right equipment, most notably a live-cell imaging chamber or incubator.
Keeping ‘em alive!
Live-cell imaging differs from conventional imaging in that you need to keep you cells alive and happy. Conventional cell-imaging protocols, which include (1) preserving your cells, (2) mounting them on a microscope slide (3) staining them, and (4) covering them with a coverslip, kills your cells (unsurprisingly!). Obviously not the best idea if you want live-cell images! For live-cell imaging you therefore need to modify your imaging protocol in a few important ways. First, you will need to forgo fixing your cells, which kills them. Second, if you choose to stain, you will need to use a stain or dye that is compatible with live cells. Third, you need a specially designed live-cell imaging chamber. Read below to learn about live-cell imaging chambers and what features you should consider when shopping for one.
Required features of your live-cell imaging chamber
Live-cell imaging chambers can vary tremendously. They can be as simple as a sealed coverslip or as complicated as a commercial perfusion chamber. But all live-cell imaging chambers should share these features:
1) Your chambers need to provide your cells with adequate media. In live-cell imaging you need to provide your cells with an optimal physiological environment. After all, you want your cells alive in LIVE-cell imaging! This means your imaging chamber must hold media.
Historically, live-cell imaging chambers were not much more than two coverslips sandwiched together with an O-ring separating them. This kind of simple chamber is adequate if you only need to image for a short period of time, say 20 minutes. But if you want to image over a longer period of time, then these simple sandwich chambers are not sufficient.
Cell media deteriorates over time and an inability to refresh your cells’ media limits the time you can image using simple chambers. Lucky for you, commercial perfusion live-cell imaging chambers let you continuously add fresh media to your cells. Thus perfusion chambers can keep your cells happier for longer, letting you image longer. However, the simplest (cheapest) perfusion chambers are casual in how they add fresh media to the cells, and thus are susceptible to the formation of chemical gradients in the culture media. So if you are serious about your cells’ long-term happiness, look for more modern (and expensive) perfusion chambers that are mindful of laminar flow.
Note: It is important that your imaging chamber does not hold too much media. For your best imaging results (read: least refraction), you want to minimize the depth of the media covering your cells.
2) Your chambers must fit on your microscope stage. Check the dimensions of your ‘scope and any potential chambers before purchasing. This would be a silly mistake to make!
3) Your cells need to be kept at the right temperature. If you plan to image for a short period of time, temperature is not a big concern, but if you plan to image for an extended period of time you need to figure out how to maintain your cells’ temperature. After all, you do not want your cells’ metabolism or signaling to change due to a poorly controlled environment! To maintain consistent temperature, you can purchase heated stages and even chambers with specialized conductive coatings.
Optional Features of Your Live-Cell Imaging Chamber
Other live-cell imaging chamber features are optional and will depend on your experimental needs. These can include:
1) Access ports. If you need to access your cells – to add regents, physically manipulate, or otherwise change your cell media during imaging – you want a perfusion chamber with ports. Commercial perfusion chambers vary a lot in price and quality. Therefore, you should carefully consider your experiment’s requirements before deciding how many and what kind of access ports your perfusion chamber needs.
2) Removable coverslips. If you wish to follow your imaging experiment with further manipulation – such as cloning, fixing or staining of your live-imaged cells – you need to be able to easily remove your coverslip to collect your cells for downstream manipulation.
So what are you waiting for? Start shopping for your new imaging chamber!