Deep ultraviolet lasers for flow cytometry

Abstract

Modern flow cytometers require multiple laser wavelengths to excite the wide variety of fluorescent probes now available for high‐dimensional analysis. Ultraviolet (UV) lasers (typically solid state 355 nm) have become a critical excitation source for the Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have pushed the number of fluorescent probes available for simultaneous analysis to nearly 30, allowing an unprecedented level of precision for immune cell analysis. However, immunologists are already seeking analyze more than 30 simultaneous parameters, requiring both new fluorochromes and corresponding laser wavelengths. A group of polymer dyes requiring deep ultraviolet (UV) excitation (~280–300 nm) is currently under development, allowing the expansion of high‐dimensional cytometry beyond the current 30 color limit. In this study, we evaluated a newly available laser emitting at 280 nm as a possible laser source for exciting these dyes. Since deep UV polymer dyes are not yet available, we used quantum nanoparticles (Qdots) as a surrogate probe to assess the utility of this laser wavelength for flow cytometry. Deep UV laser light was found to excite Qdots as well as traditional UV sources. Deep UV 280 nm did not excite BUV dyes well, suggesting that BUV and deep UV polymers will be spectrally compatible with low crossbeam spillover issues. Deep UV excitation did excite considerable autofluorescence in the violet to blue range, a limitation that will need to guide deep UV fluorochrome development. A deep UV 280 nm laser may therefore be the next essential wavelength for high‐dimensional flow cytometry. © 2018 International Society for Advancement of Cytometry

Deep ultraviolet 266 nm laser excitation for flow cytometry

“…Autofluorescence excitation at 320 nm has been previously observed to be higher than at UV 355 nm, reducing sensitivity for short wavelength fluorochromes like BUV395 by almost half [6]. Excitation at 280 nm generated far more cellular autofluorescence, causing BUV395 to be unusable at this excitation wavelength [8]. Cellular autofluorescence therefore has a significant impact of fluorochrome detection, increasing backgrounds and reducing instrument sensitivity for fluorochromes emitting in the 380-500 nm range.…”

“…Cellular autofluorescence therefore has a significant impact of fluorochrome detection, increasing backgrounds and reducing instrument sensitivity for fluorochromes emitting in the 380-500 nm range. Above this emission wavelength, autofluorescence is much lower [6,8]. To determine the level of cellular autofluorescence at DUV 266 nm, unlabeled human PBMCs were analyzed using a range of bandpass filters with the laser both on and off.…”

Spectrally encoded microspheres for immunofluorescence analysis

“…117 In 2017, there were more than a dozen of flow cytometers with the option of three-laser excitation (405, 488, and 633 nm) for more than eight measurements of fluorescent dyes. 118 Because recently developed polymer fluorochromes also require deep UV excitation (280-300 nm), 36 UV lasers have become a critical excitation source for the Brilliant UV series. π-conjugated polymers act as collections of optical subunits, which can respond cooperatively in various energy transfer and/or quenching processes.…”

“…Despite some drawbacks like the autofluorescence or even disruption of organic molecules initiated by UV irradiation, UV lasers become useful for certain types of analysis. To overcome these obstacles, one can use a multicolor analysis with UV dyes, which has negligible spillover into other detectors, or employing quantum dots (QDs) as biomarkers nonsusceptible to degradation upon UV irradiation 36 …”

“…To overcome these obstacles, one can use a multicolor analysis with UV dyes, which has negligible spillover into other detectors, or employing quantum dots (QDs) as biomarkers nonsusceptible to degradation upon UV irradiation. 36 In the same way, the sensitivity of semiconductor detectors has dramatically increased with a corresponding price drop during recent years. Unsurprisingly, the device developers and manufacturers follow this trend, and some new flow cytometers models are now equipped with reliable and shock resistant APD rather than PMT.…”