Conventional lasers do the job for cytometry

Conventional lasers do the job for cytometry

I write with regard to the article, “Semiconductor lasers shed new light on flow cytometry” (August, p. 69), which described semiconductor lasers for flow cytometry. On the whole I found the ­article informative, presenting a succinct and fairly comprehensive overview of the technology and where it is going.

The article might have been improved had the focus remained more on the topic of flow cytometry itself rather than discussing Coherent’s and Novalux’s VECSEL technologies. While their contribution is indeed important, this seemingly intentional glorification of a single semiconductor-laser technology, a topic already ­covered by many other articles in the technical press, played down the fact that ­conventional semiconductor-laser technology is equally capable of ­providing the required performance, ­albeit at other wavelengths.

The cusp of the article, at least in terms of laser performance, might have perhaps placed more focus on the ­requirement for a Gaussian beam. A gauge of laser performance in flow ­cytometry is the ability to accurately sort standard reference beads, the figure of merit being termed “coefficient of variance,” or CV. A once misquoted specification was that a laser beam should be “Gaussian to within 1%” to ensure good CV results. Ignoring for the moment the issue of whether beam profilers measure to this level of accuracy, I suspect very few lasers actually achieve this level of perfection. On pursuing the origins of this ­requirement, what was really meant was that the CV value generated should be 1%-a value widely regarded as ­excellent but actually only necessary for specific ­cytometric applications.

As it turns out, a clean near-Gaussian beam, as seen from a gas laser, is currently the favored beam profile simply because the associated CV can be this good, which results in the ability to differentiate between very similar particles.

An application mentioned in the ­article, livestock sex selection, is a very good example for which this differentiation is crucial; sperm sorting is achieved via DNA identification, in which the difference between the male and female DNA as seen with flow ­cytometry methods is very slight. As far as I am aware, the VECSEL technology highlighted in the article is not used here, as other factors come in to play.

Toptica Photonics, and I suspect others, are working with the industry to provide lasers, for example at UV and violet wavelengths, incorporating conventional laser diodes either selected for near-Gaussian or spatially filtered for near-perfect Gaussian beam quality. It should be noted, however, that a short examination of the interaction space between the laser light and the particles, and the appreciation of the varying paths through the Gaussian beam, quickly leads one to the conclusion that intentionally modified beam profiles might serve this application much better.

Several semiconductor-laser technologies will be required from the ­market in the long term, primarily ­because the assorted fluorescence tags used for generation of side scatter require different excitation energies. These lasers offer compact size, efficiency, and simplicity, increasing reliability and performance, and ­reducing cost of ownership-all of which make ­semiconductor-laser technology ­increasingly attractive for life sciences in general.

Gregory Flinn
Toptica Photonics
Munich, Germany

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