Experiment information
For imaging, cell samples must be chemically fixed and highly optimised to remove all fluorescence background. High background fluorescence or strong optical aberrations will cause MINFLUX measurements to fail. This means that measurements are restricted to within a few µm of the coverslip and while MINFLUX imaging of tissue in possible in principle, careful sample preparation is required. 3D measurements are more sensitive to background and aberrations than 2D.
The photo-switching behaviour of cyanine dyes breaks down when dye molecules in a sample are closer than 10nm, and switching is uncontrollable when labelling density is too high. In both situations, we recommend using DNA-PAINT to achieve blinking instead.
For tracking, MINFLUX generally works well with samples that have already been optimised for camera based single molecule tracking although further optimisation to reduce background or labelling concentration is sometimes required. Imaging with DNA-PAINT/photoactivatable dyes a secondary context label that can be seen in the 488nm confocal channel is necessary.
Recommended fluorescent probes:
| MINFLUX technique | Recommended dye(s) |
| Localisation microscopy with ONE photo-switching Cyanine dye | Alexa 647, Abberior Flux 647 |
| Localisation microscopy with TWO photo-switching Cyanine dyes | Abberior FLUX 640 and Abberior FLUX 680 |
| Localisation microscopy with DNA-PAINT | Atto655 |
| MINFLUX tracking | Atto 647N, JFX650 |
