Sequential imaging involves exciting fluorophores on the specimen with only one laser at a time and collecting fluorescence photons emitted by the excited fluorophores. Then, by swapping to another laser wavelength and detecting photons emitted from another fluorophore, spectrally separated signals can be collected. Provided each laser line excites only one fluorophore, all emitted photons will be derived only from the relevant fluorophore with no spectral bleedthrough. When sequentially imaged a specimen that has been dual labelled with both DAPI and AF488, a single laser is used for excitation and the emitted photons collected (image A). Then the other laser is used for excitation and the relevant emission photons collected (image B).

Image A

Sequential imaging of DAPI and AF488 in a dual labelled specimen. In this example, DAPI is imaged first (and the 488nm laser is turned off during image collection).

Image B

Sequential imaging of DAPI and AF488 in a dual labelled specimen. When imaging the second fluorophore (AF488), the 405nm laser is turned off during image collection and the only signal collected is derived from AF488.

Simultaneous imaging three or four fluorophores in the same specimen will almost always produce significant spectral bleedthrough (as can be seen below). Careful selection of fluorophores combined with sequential imaging can usually eliminate (or at least minimise) spectral bleedthrough.

Sequential imaging of DAPI, AF488, AF568 and AF647 in a four fluorophore labelled specimen can minimise spectral bleedthrough.

Sequential imaging has the advantage of minimising spectral bleedthrough and, in most cases, should always be used when performing analyses of colocalisation of multiple fluorophores. However, sequential imaging of four fluorophores means image collection time will take at least four times longer. Additionally, the temporal separation between each fluorophore image using sequential imaging usually precludes using this technique to image rapidly moving live samples.

Some Possible Fluorophore Combinations

1. DAPI bleedthrough into AF488 channel.
2. DAPI bleedthrough into AF568 channel if DAPI signal is very strong.
3. DAPI bleedthrough into AF488 channel and, if DAPI signal is very strong, also into AF568 channel.
4. DAPI bleedthrough into AF488 channel but not AF647 channel.
5. Minimal DAPI bleedthrough into AF568 channel; significant AF568 bleedthrough into AF647 channel.
6. Spectral bleedthrough into all channels except DAPI channel.