Laser Doppler velocimetry for particle size determination by light scatter within the sheath flow cuvette

Zarrin, Fahimeh; Bornhop, Darryl J.; Dovic̀hi, Norman J.

doi:10.1021/ac00133a015

Cited by 21 publications

(9 citation statements)

References 37 publications

(20 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1A). The time-dependent, conventional two-color green and red fluorescence detector emission signals from phagocytosed GY microspheres [v(t) Gr ] and PI [v(t) Red ] are Gaussian-shaped pulses that result from passage of the cell across the laser beam, which can be expressed in approximate form as v(t) Gr ϭ V GM · e Ϫa 2 (tϪt0) 2 and v(t) Red ϭ V PI · e Ϫa 2 (tϪt0) 2 , (1) where V GM and V PI are the maximal green and red fluorescence signal intensities, respectively, t is time, and a is a term related to the laser beam height and to the velocity of a cell crossing the laser beam at time t 0 (29). However, if there is signal cross-talk between the fluorescence measurement channels due to overlapping emission spectra, such as from the fluorescence from phagocytosed microspheres interfering in the red fluorescence channel, then equations 1 can be rewritten as:…”

Section: Materials and Methods Theorymentioning

confidence: 99%

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

2000

View full text Add to dashboard Cite

Section: Materials and Methods Theorymentioning

confidence: 99%

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

2000

View full text Add to dashboard Cite

“…where V is the fluorescence signal intensity, w is the angular excitation frequency, 4 and m are the signal phase shift and demodulation terms (6) associated with a single fluorescence decay time, t is time, and a is a term that relates the velocity of the cell across the laser beam at time t, (21). Equation (1) is derived for cells that are excited by an excitation source with 100% modulation.…”

Section: Methodsmentioning

confidence: 99%

Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry

Steinkamp

Crissman

1993

Cytometry

View full text Add to dashboard Cite

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a longpass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PIand FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by m/2 + degrees and the phase of the other detector channel reference signal by -m/ 2 + +z degrees, where and +z are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering. Key terms: Fluorescence lifetime, phasesensitive detection, propidium iodide, fluorescein isothiocyanate, fluorescence phase-resolved measurement Flow cytometry is a n important clinical diagnostic tool for use in immunology, hematology, and oncology, including its application to basic biomedical research. Clinical tests and biological experiments often require the labeling of cells with multiple fluorochromes for correlated analysis of cellular properties. A major limitation of these procedures is the availability of fluorescent dyes with a common excitation region, i.e., requiring only one excitation source, and emission spectra that are sufficiently separated to permit measurement by multicolor detection methods (18) that employ dichroic and bandpass filters. To alleviate the problem, multiple excitation sources are usually employed (17) to excite sequentially cells labeled with fluorochromes that have separated excitation spectra. This approach has greatly increased the number of fluorochromes available for multicolor labeling studies, but the instrumentation has become increasingly complex in both design and function.We have developed a new flow cytometric method (19) to separate signals from fluorochromes by phaseresolved measurement of fluorescence emission signals. Our intention is to resolve signals from fluorochromes with overlapping emission spectra, but 'Research reported in this article was performed under the auspices of the U.S. Department of Energy.

show abstract

“…The detection volume was small (60 fl) and the flow velocity was slow (60 cm/s). Dovichi and colleagues (13) studied the light scatter of submicrometer particles within a sheath flow cuvette using a helium-cadmium laser with Doppler velocimetry and detected 0.090-µm diameter polystyrene beads. Although the microscope-based jet-onopen-surface cytometer invented by Steen and co-workers has been shown to have high scatter sensitivity, Steen (14) only reported a SSC sensitivity of 0.2-µm polystyrene beads.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer

Mariella¹,

Huang²,

Langlois³

1999

Cytometry

View full text Add to dashboard Cite

Extracellular vesicles (EVs) are commonly studied by flow cytometry. Due to their small size and low refractive index, the scatter intensity of most EVs is below the detection limit of common flow cytometers. Here, we aim to improve forward scatter (FSC) and side scatter (SSC) sensitivity of a common flow cytometer to detect single 100 nm EVs. The effects of the optical and fluidics configuration on scatter sensitivity of a FACSCanto (Becton Dickinson) were evaluated by the separation index (SI) and robust coefficient of variation (rCV) of polystyrene beads (BioCytex). Improvement is defined as increased SI and/or reduced rCV. Changing the obscuration bar improved the rCV 1.9-fold for FSC. A 10-fold increase in laser power improved the SI 19-fold for FSC and 4.4-fold for SSC, whereas the rCV worsened 0.8-fold and improved 1.5-fold, respectively. Confocalization worsened the SI 1.2-fold for FSC, and improved the SI 5.1-fold for SSC, while the rCV improved 1.1-fold and worsened 1.5-fold, respectively. Replacing the FSC photodiode with a photomultiplier tube improved the SI 66-fold and rCV 4.2-fold. A 2-fold reduction in sample stream width improved both SI and rCV for FSC by 1.8-fold, and for SSC by 1.3-and 2.2-fold, respectively. Decreasing the sample flow velocity worsened rCVs. Decreasing the flow channel dimensions and the pore size of the sheath filter did not substantially change the SI or rCV. Using the optimal optical configuration and fluidics settings, the SI improved 3.8Á10 4 -fold on FSC and 30-fold on SSC, resulting in estimated detection limits for EVs (assuming a refractive index of 1.40) of 246 and 91 nm on FSC and SSC, respectively. Although a 50-fold improvement on FSC is still necessary, these adaptions have produced an operator-friendly, high-throughput flow cytometer with a high sensitivity on both SSC and FSC.

show abstract

Laser Doppler velocimetry for particle size determination by light scatter within the sheath flow cuvette

Cited by 21 publications

References 37 publications

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry

Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer

Contact Info

Product

Resources

About