Sort Calculator by Frequency

Rmax is an estimate of sort recovery influenced by several factors including;

1) How evenly illuminated the particles are through the laser beam,

2) How discrete the populations appear in the scatter plots,

3) How well the electronics are dealing with the identification and sorting of a target population in a sample,

4) How well prepared the sample is, ie., the sample is mono-dispersed.

The Centre Stream Catch (CSC) is a sample of the waste stream during a sort. Any target particle not sorted will end up in the waste stream.

Rmax# uses the number of target and non-target particles in both the original and the CSC and it is recommended if the frequency of target particles are greater than 5%. It is defined as:

Where O_t and O_nt are target and non-target in the original sample whereas C_t/C_nt are target and non-target in the Centre Stream Catch.

Data Collection

Set up a sort Target(s) only one-drop sort mode. Sort beads made up of blank beads (50%) and target fluorescent beads (50%). The beads should be mostly single particles.

Run the beads at an event rate about 1/10 to 1/20 the drop generation rate.

Sort the target and at the same time collect the CSC. Fill a 5ml tube with the CSC and centrifuge. Decant the supernatant to leave about 500µl, analyse, and record the Target%.

Enter the original Target and non-target values and the CSC Target and non-Target values in the calculator. To compare an experimental sample's Rmax with the beads, run some of the sample at the same rate of the beads and collect the CSC.

Results

The higher the Rmax the more Poisson like the particles behaviour. Run the beads first to establish how well the instrument is performing by assuming that they are behaving in an idealised Poisson way.

If the Rmax is low (less than 90%) then investigate the instrument for instabilities, such as fluidic instabilities or electronic issues with the sort or an incorrect drop delay etc.

If the Rmax is 90% or higher, then run the experimental sample.

Calculating Rmax on the experiment evaluates whether the particles are behaving in a Poisson way, ie., that the sample is a single particle suspension.

If the Rmax is low compared to the bead Rmax look into the sample’s preparation to reduce particle clumping

Rmax can also be used to set the optimal drop delay. Set the initial drop delay and collect the CSC. Alter the delay by small steps (fractions of a drop) and collect the CSC. The delay with the highest Rmax value is optimal drop delay