Hi there! Here is a question that confused me: if the resolution of flow cytometry is 1 um, then why are 100 nm calibration beads still detected well (according to the events on the dot plots)? I thought 1 um is the smallest size flow cytometer can detect.
Hi Michelle
This depends greatly on the FACS machine you have. For conventional flow cytometry the limit of detection is around 500nm.
What you mean when you say that 100 nm calibration beads are still detected well? Theorycally , under 488 nm (the wave length of the blue laser used to define the FSC SSC) you have not a good resolution of beads under this diameter size. This mean that (for the most powerful flow cytometer available) you can not create a scale with calibrating bead under 488 nm even if you can distinguish different populations. The size will be not accurately defined.
Hey guys,
to my knowledge the smallest calibration beads available have a diameter of around 2.5 µm (microns!). So, sharing Matthieu's confusion, I'm not sure how 100 nm (NANOmeter) calibration beads fit into the picture. These are values at the wavelength scale, not diameter scale.
I think the closest thing to the concept of FSC 'resolution' is rather the size range within which events can be detected. As Matthieu said, this strongly depends on the machine used, but e.g. you can't reliably detect bacteria on a BD LSRII or FACSCanto, which however is easily possible on an Accuri machine since the latter has a greater detection range at the lower end of the 'size scale'.
Even when using particles that have a defined diameter (such as beads), the corresponding FSC-A value of these can not be used to reliably draw conclusions about the actual size of cellular events since their morphological properties (and hence their FSC-A values) very strongly depend on the treatment etc.
A good example for this is, when you fix and permeabilize cells they appear 'smaller' (lower FSC-A value) than unfixed cells even though in reality they don't actually shrink. If anything they become less compact due to the permeabilization step.
I have already used beads of around 90 nm. But as you said Eric, the refractive index between beads and cells is different due to morphological and physical propoerties.
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