#red dyes

[affreca]

Dye sampling continues. Over the course of the week I got through the blues and purples. Today was a work from home day, so I could get three batches of reds and pinks dyed. I will take proper comparison photos when I have time in sunlight.

[biolegend]

Getting Acquainted with Red Dyes

Have you ever wondered why there are so few options for the red laser on your flow cytometer? I think about this a lot and figure you may be wondering the same, so I decided to open the discussion here in our blog. Considering the wide spectrum of dyes for other lasers, to me the question is more out of curiosity than utility. I mean, given the many choices available from some other lasers, perhaps there is not such an excruciating need now for these dyes. However, even before the introduction of such a wide variety of dyes (in the violet laser for example), there were very few dyes available for the red laser. So, why is this? I have my own opinion, which I will discuss in brief with you. I will also share the thoughts of a few very capable people in the fields of flow cytometry and chemistry. Knowing how cytometry works, we can see that we will have more room to find molecules that are excited by the low end of the spectrum (violet laser) and emit at the higher end. This is in contrast with molecules excited by the high end of the spectrum (a red laser) that also have to emit in the high end of the spectrum: Cheryl Kim, the core manager at the La Jolla Immunology and Allergy Institute, agrees with this reasoning. In addition, she points out the problem of intra-laser compensation, which is a good point. Prof. John Nolan from La Jolla Bioengineering Institute believes that the lack of dyes for the red laser is probably due to low demand for red excited fluors, which in turn is caused by the poor sensitivity of the detectors in the red zone. A potential solution for this could be the detection of the emitted fluorescence with sensors other than the photomultiplier tubes (PMT) used in conventional flow cytometers. An alternative could be to use a Charge-Couple Device (CCD), such as the ones used in digital photography, and apply it to spectral flow cytometry. In addition to this, our fluorescence chemistry expert, Kelly Lundsten, highlights that there are certain chemical properties of fluorescent dyes that also restrain the development of molecules capable of emitting in the infrared. Fluorophores in that spectral range are large at 1kD-3kD (compared to about 500D for FITC). As they grow larger, they become more hydrophobic, more susceptible to photobleaching and inefficient at producing fluorescence per photon absorbed. Dyes for the red laser also require big electronic clouds to tune the spectra, and this is not optimal for biological applications: Structures taken from: http://www.atdbio.com/content/34/Alexa-dyes So, do you have any other ideas or suggestions on why there are so few dyes for the red laser in conventional flow cytometry? Contact us here.