A comparison of avalanche photodiode and photomultiplier tube detectors for flow cytometry

Lawrence, W. G.; Váradi, Gyula; Entine, G.; Podniesinski, Edward; Wallace, Paul K.

doi:10.1117/12.758958

Cited by 22 publications

(20 citation statements)

References 12 publications

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“…The former showed greater resolution of signal from background for the fluorophores on the conventional system, while for the latter we found a decrease in signal spread from measurements made on the spectral system, compared to the conventional system, and in many cases, this was accompanied by an increase in resolution of signal from spread. It is likely that the use of avalanche photodiode (APD) detectors on the spectral system contributes to the improved resolution of some of these signals, compared to the photomultiplier tube (PMT) detectors used on the conventional system, as APD have been shown to perform better in spectral regions over 650 nm due to increased sensitivity in these parts of the spectrum (23). Importantly, although the resolution of individual signals from background was lower on the spectral system compared to the conventional system in this comparison, the improved signal resolution from signal spread in most cases indicates better performance in a mixed panel context.…”

Section: Signal Comparability Of the Systemsmentioning

confidence: 99%

Evaluating spectral cytometry for immune profiling in viral disease

Niewold¹,

Ashhurst

Smith

et al. 2020

Cytometry Pt A

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In conventional fluorescence cytometry, each fluorophore present in a panel is measured in a target detector, through the use of wide band-pass optical filters. In contrast, spectral cytometry uses a large number of detectors with narrow band-pass filters to measure a fluorophore's signal across the spectrum, creating a more detailed fluorescent signature for each fluorophore. The spectral approach shows promise in adding flexibility to panel design and improving the measurement of fluorescent signal. However, few comparisons between conventional and spectral systems have been reported to date. We therefore sought to compare a modern conventional cytometry system with a modern spectral system, and to assess the quality of resulting datasets from the point of view of a flow cytometry user. Signal intensity, spread, and resolution were compared between the systems. Subsequently, the different methods of separating fluorophore signals were compared, where compensation mathematically separates multiple overlapping fluorophores and unmixing relies on creating a detailed fluorescent signature across the spectrum to separate the fluorophores. Within the spectral data set, signal spread and resolution were comparable between compensation and unmixing. However, for some highly overlapping fluorophores, unmixing resolved the two fluorescence signals where compensation did not. Finally, data from mid-to largesize panels were acquired and were found to have comparable resolution for many fluorophores on both instruments, but reduced levels of spreading error on our spectral system improved signal resolution for a number of fluorophores, compared with our conventional system. Furthermore, autofluorescence extraction on the spectral system allowed for greater population resolution in highly autofluorescent samples. Overall, the implementation of a spectral cytometry approach resulted in data that are comparable to that generated on conventional systems, with a number of potential advantages afforded by the larger number of detectors, and the integration of the spectral unmixing approach.

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Section: Signal Comparability Of the Systemsmentioning

confidence: 99%

Evaluating spectral cytometry for immune profiling in viral disease

Niewold¹,

Ashhurst

Smith

et al. 2020

Cytometry Pt A

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“…Improving upon the sensitivity of conventional flow cytometers, we have developed a semiconductor-based flow cytometer, called the CytoFLEX, which pairs silicon avalanche photodiodes (APDs) with wavelength-division multiplexing (WDM), an optimized flow-cell design, and diode lasers in order to maximize signal and minimize noise. Silicon APDs are semiconductor photodetectors that have a higher quantum efficiency and lower electronic noise than traditional photomultiplier tubes, resulting in increased light-detection sensitivity across a larger wavelength range 13–15 . The WDM design, adapted from fiber-optic technology used in the telecommunications industry, eliminates the dichroic mirrors that are traditionally used to divide light into color bands within filter trees, preventing the 20–50+% signal losses that occurs in a typical flow cytometer prior to even reaching the bandpass filters (Fig.…”

Section: Introductionmentioning

confidence: 99%

A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles

Brittain

Chen

Martinez

et al. 2019

Sci Rep

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The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

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“…Importantly, single photons can be detected with PMTs, but discrimination of single versus multiple photons is difficult. Avalanche photodiodes have better QE and better sensitivity in the green and red region of the visible spectrum compared to PMTs [64].…”

Section: Photomultiplier Tubes (Pmts)mentioning

confidence: 99%

Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies

Youker¹

2018

Photon Counting - Fundamentals and Applications

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The resolution of light microscopy was thought to be limited to 250-300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.

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A comparison of avalanche photodiode and photomultiplier tube detectors for flow cytometry

Cited by 22 publications

References 12 publications

Evaluating spectral cytometry for immune profiling in viral disease

Evaluating spectral cytometry for immune profiling in viral disease

A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles

Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies

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