AccuCount Rainbow fluoreszierende Partikel

Analysis of single nanoparticles by flow cytometry involves the measurement of weak signals that are close to the instrument’s detection limits. Distinguishing the signal from particles of interest from background particles in buffers and from optical and electronic noise can be difficult and requires careful consideration of the measurement approach, control experiments, and examination of the resulting data. 

In applying this check, we have identified an artifact resulting from inappropriate trigger channel threshold selection that may not be obvious to the casual user. When measuring weak signals near noise or background levels, it is intuitive and common for the operator to adjust the trigger threshold to minimize the „false triggers“ detected by the system, and then analyze the unknown sample. Interpreting the events detected over the background as single particle measurements. We show here that when using this approach to measure particles whose signals fall below the trigger threshold, only coincident events are detected, leading to erroneous measurements of both particle number and brightness. We propose that in many cases the analysis of weak nanoparticles is best achieved using a fluorescence channel as a trigger.

introduction

There is significant interest in using flow cytometry for multiparameter analysis of small biological particles such as B. cell-derived microvesicles (including ectosomes and exosomes), as well as bacteria, viruses and intracellular organelles. 

  • However, the development of validated protocols for this purpose has proven to be challenging, as the results are strongly influenced by variables of pre-analytical sample preparatio  by variations in staining, measurement and analysis procedures, and by differences in instrument performance between different makes and models of flow cytometers and even between the same instrument model. 
  • All of these factors are the subject of active investigations and discussions, which have raised awareness of all the experimental details that can affect the results. We’ve identified one particularly subtle but ubiquitous artifact that we believe deserves attention.
  • In flow cytometry of cells, light scatter, generally a forward angle, is often used to trigger the data acquisition system to take a measurement. Light scatter from cells is usually well resolved from background and there is usually little ambiguity as to the nature of the events detected.
  • As the particle size (diameter) decreases, the light scattering intensity decreases nonlinearly and with measurement angle dependence , so for very small particles the light scattering intensity may not be resolved by random small particles in the shell or sample solution, or by optical or electronic noise of the instrument.
  • The light scattering response of a flow cytometer has often been characterized with uniform polymer beads, leading to the realization that different instruments with different light collection angles perform differently and that side scatter generally has better resolution than forward scatter due to the lower levels of background light scatter orthogonal to the excitation beam.
  • Such studies have also led to the finding that polymer beads are unsuitable as size calibration particles for application to membrane vesicles, as the latter have a lower refractive index, ). Therefore, current best practice seems to be to use polymer beads as reference particles to standardize instrument setup rather than as size standards for calibration.
  • But even when these factors affecting the measurement are recognized, there is a measurement artifact that occurs when light scattering is used to trigger the detection of particles whose light scattering is below the instrument’s noise threshold and whose origin is random. We illustrate this artifact and suggest that fluorescence triggering may be more appropriate for measuring small, dark particles.

Materials and methods

Nile Red microspheres (0.53 µm diameter and 0.11 µm diameter) were from Spherotech. Working solutions of approximately 1 × 10  8  particles/mL were made up in 0.1 μm filtered nanopure water using the mass concentration provided by the vendor and the diameter and density of the particles according to the relationship N = (6 W/3.14 × P × D  3  ) manufactured. × 10  12  particles, where W is the weight of the polymer in grams, P is the polymer density (1.05 for polystyrene), and D is the particle diameter.

Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences) equipped with a standard laser and filters, using 0.1 µm filtered nanopure water as the sheath fluid. Each sample was run for 60 seconds at a low sample flow, which was determined to be 0.124 µl/second, measured atUse of Accucount counting beads (Spherotech). Samples were measured using either light scatter (FALS plus SSC) or fluorescence (FL2 channel: 488 nm excitation, 585/42 nm bandpass)) triggering and data analyzed using FCS Express 4 (De Novo Software). The fluorescence intensity axis was calibrated using 8-tip Rainbow beads (Spherotech) that had been cross-calibrated with Quantabrite PE beads (BD Biosciences) on this instrument. Data files were deposited in the Flow Cytometry Data Repository (flowrepository.org).

results and discussion

Measuring small, faint particles often requires working close to the instrument’s noise floor. When detectors are operated at high gain, a sample of the filtered buffer will generate spurious events resulting from electronic or optical noise or signals from particles smaller than the pore size of the filter used.  In typical use, trigger thresholds are set to minimize the frequency of such background events to a minimum level, after which the sample is measured and the data analyzed and reported. However, if care is not taken to perform some necessary control experiments, the reported results may be incorrect. It is instructive to illustrate this issue with a well-understood probe such as fluorescent microspheres.

We used two commercially available fluorescent polymer microspheres with diameters of 530 nm and 110 nm and analyzed them at different particle concentrations using either light scattering or fluorescence channels to trigger detection. A sample of the filtered buffer was used to set the trigger threshold at such a level that the „noise“ event frequency was ~10/second.

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As shown, the 530 nm particle is readily detected in both light scattering and fluorescence channels and using a dual channel trigger threshold (FSC and SSC) or a single channel trigger threshold (fluorescence) (grey dashed lines). Individual beads are clearly visible in both the side scatter and orange fluorescence channels, yielding an absolute fluorescence intensity value of 4900 MESF-PE with both triggering methods. Coincident events that are approximately twice as bright are also easily identified in both the SSC and fluorescence channels. Neither the light scatter nor the fluorescence distributions change when the sample is diluted, although it is evident that the fluorescence signals are more clearly resolved from background than the light scatter signals.

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