It all depends on the available lasers & pmt filters on your microscope and the fluorochromes/flourescent proteins that you can use. Is this in a culture dish? Can you use a uv or Violet laser, or would that kill your cells? How fast do your acquisitions need to be? If you can tolerate slower imaging, then you can line/frame shift between multiple lasers to increase the number of possible channels. A good tool for figuring this out is BD's spectrum viewer (just Google it for link). The spectrum viewer is meant for flow cytometry, but it is great for looking at spectral overlap.

Generally if you are not an expert with microscopy and don't have access to specialized microscope, you are going to have difficulty trying to image more than 3 channels for live cells. Two photon generally is not compatible with high level fluorochrome multiplexing.

Are you able to use fluorochromes or only flourescent proteins? If only fluorescent proteins, this will be very difficult to even achieve 3 channels.

Can you acquire multiple channels off a single laser? If not you won't be able to do this. Most microscopes are not made to acquire more than 3 or 4 channels total.

For instance, if you can use fluorochromes and your microscope is capable you could acquire 3 to 4 channels off a uv or Violet laser and an additional 3 channels off the blue/green laser by using line/frame switching. Or you could acquire 3 channels off your 488 and 1 (maybe 2) off your 633. Many of the bright and narrow range fluorochromes bleach quickly and are primarily used for cytometry.

I might consider trying to use wide field flourescent microscopy for this, and try to limit your channels to 3 colors. You can use morphology to help distinguish between some cell types. With microscopy you can't have it all. If you want lots of channels, you have to sacrifice imaging speed; if you want fast imaging you have to sacrifice channels, resolution, or 3d capability, etc. Light sheet microscopy can actually accomplish what you want to do, but to my knowledge nobody has yet built a light sheet scope with high multiplexing capability.

Hi Roselien,

I agree more or less with Daniel. I would also start up with three channels, a fourth one should not be such a big problem. I would start with a simple set-up and add more complexity and colors when you have acquired the first Images.

Kind regards

Soenke

On a spinning disk confocal, if you have two different cameras to detect different wavelengths then you could do two colors simultaneously. Otherwise, you could do one color at a time with one camera, so like snap n number of channels and you could merge them together once acquired.

It's always the best if you could do two colors simultaneously then you are basically avoiding a lot of lasers to hit your sample for a longer time (which otherwise might bleach your sample).

If you are doing one channel at a time (especially if you have live samples) then I'd prefer to do the exposure time as short as possible, laser as low as possible.

Thank you all!