Rainbow Particles...

Are useful for ensuring your flow cytometer’s lasers are properly calibrated and aligned. 

First and foremost, it should be noted that Rainbow Particles are not compensation beads, nor can they be used to estimate cell size/number. Rainbow Particles are designed to ensure a flow cytometer is properly calibrated, standardize instruments for longitudinal studies, and evaluate new instrument hardware (e.g., bandpass filters, photomultiplier tubes (PMTs) and lasers). They are excited by any wavelength in the range of 365-650 nm, which includes the violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (633 nm) lasers. They emit in all channels off of those lasers, but at varying intensities.  Rainbow beads can be found in 1 peak, 6 peak and 8 peak options and help characterize the range of sensitivity and linearity of the instrument PMTs and lasers. Rainbow Particles are not compatible with the UV laser.


When you excite fluorophores conjugated to antibodies bound to your cell, you want to ensure that they fall within an ideal range of performance for each laser’s PMT positions. This can be monitored by using the particles and plotting a channel from one laser against a channel from the other laser.

 

 

In the “bad” alignment plot, the violet laser may be underpowered or improperly aligned.

 

As mentioned previously, we offer one, six, and eight peak options, each of which can aid with longitudinal studies to check whether peaks routinely show up at the same fluorescent intensity using a calculation of staining index. For the one peak choices, there are mid-range and  bright intensity peaks. The mid-range option can help represent antigens of moderate expression, while the bright intensity option is ideal for highly expressed targets. Six or eight peak options offer a larger variety of fluorescent intensities. While six peaks is typically enough for most users, the eight peak option can be selected if more sensitivity is desired or the linear range of the PMT or photodiode is expanded.

For more information on the use of calibration beads, take a look at this article by Perfetto, et al.