Trends and Developments in Flow Cytometry Instrumentation

Shapiro, Howard M.

doi:10.1111/j.1749-6632.1993.tb38774.x

Cited by 22 publications

(7 citation statements)

References 29 publications

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“…Diode and DPSS laser sources are becoming common fixtures in flow cytometers (4,16,17); as the available wavelengths increase, they should find many applications both in specialized fluorescence applications and in more routine applications (such as the ones illustrated in this study) as improved excitation sources for.…”

Section: Discussionmentioning

confidence: 99%

DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry

et al. 2005

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IntroductionBlue‐green 488‐nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less‐than‐optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium–neons and, more recently, diode‐pumped solid state (DPSS) 532‐nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561‐nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity.MethodsA DPSS 561‐nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R‐ and B‐forms of PE were analyzed and compared with conventional 488‐nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green‐yellow excited low molecular weight fluorochromes, and a rhodamine‐based caspase substrate. Additionally, cells labeled with PE and co‐labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible.ResultsThe DPSS 561‐nm laser gave a several‐fold improvement in the fluorochrome to autofluorescence ratios between PE‐labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561‐nm source gave a 6–7‐fold improvement in sensitivity over 488‐nm excitation, and gave excellent excitation of yellow‐green excited fluorochromes and rhodamine‐based physiological probes. Yellow‐green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation.ConclusionsDPSS 561‐nm laser excitation gave significantly improved sensitivity for both PE‐labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector. Published 2005 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry

et al. 2005

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“…Flow cytometers have seen a recent dramatic increase in the number of simultaneous fluorochromes they can analyze. Single and dual laser flow cytometers have given way to instruments with five or more laser wavelengths, allowing excitation of a wide variety of fluorescent probes . Modern instruments capable of high‐dimensional analysis (10 or more simultaneous fluorochromes) are typically equipped with solid‐state lasers emitting at 488 nm (cyan), 633–640 nm (red), 532 or 561 nm (green or yellow), and 405 nm (violet).…”

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confidence: 99%

Deep ultraviolet lasers for flow cytometry

Telford

Georges²,

Miller

et al. 2018

Cytometry Pt A

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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

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“…These additional wavelengths give access to a larger array of fluorescent probes than a single laser wavelength alone. Red lasers in particular allow detection of many long red and NIR fluorescent probes, including phycobiliprotein probes like allophycocyanin (APC), and cyanine dyes and their many derivatives [ 3 , 4 , 5 ]. As with imaging, flow cytometric analysis with red lasers benefits from high sensitivity and low cellular autofluorescence.…”

Section: Introductionmentioning

confidence: 99%

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

et al. 2015

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Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

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Trends and Developments in Flow Cytometry Instrumentation

Cited by 22 publications

References 29 publications

DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry

DPSS yellow‐green 561‐nm lasers for improved fluorochrome detection by flow cytometry

Deep ultraviolet lasers for flow cytometry

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

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