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.