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Sub-micrometer Particle Standards for Quantitative Flow Cytometry
Interlaboratory Comparison of Number Concentration Measurements
The number concentrations of both unstained and fluorescently-labeled polystyrene sub-micrometer sphere suspensions with nominal diameters from 100 nm to 500 nm have been measured using seven different techniques. Number concentration values that agree within the known uncertainties of the instruments are considered high accuracy—typically within 10% of each other. Only three of the techniques were able to achieve this level of agreement. A consensus-based protocol using these three high-accuracy, validated techniques has been developed at NIST to certify sub-micrometer particle suspensions for number concentration.
Consensus techniques used for certification of number concentration:
- Dry mass with transmission electron microscopy (DM)
- Flow cytometery (FCM)
- Fluorescence microscopy with charged slide (FM)
Instrument Calibration using Certified Reference Beads
The value-assigned suspensions described above are inteded for use as new reference materials for the calibration of instruments capable of sub-micrometer particle number concentration measurements. NIST has demonstrated the use of such reference beads for number concentration calibration of flow cytometers (see below) and for other techniques, including particle tracking analysis (PTA), microfluidic resistive pulse sensing (MRPS), and asymmetric flow field fractionation with multi-angle light scattering (AF4-MALS).
ERF Fluorescence Intensity Assignment of Calibration Beads
NIST assigns equivalent reference fluorophores (ERF) units to the fluorescence intensity of a calibration bead by using a reference fluorophore solution as a standard light bulb. The integrated intensity of the emission spectrum of a reference fluorophore solution is compared to the integrated intensity of the emission spectrum of a bead solution. The fluorophore concentration of the reference fluorophore is known, allowing fluorescence intensity to be quantified in terms of fluorophore concentration, i.e., ERF units.
The number concentration of the bead suspension is determined using the consensus technique values as explained above. The fluorescence intensity and number concentration values of the bead suspension are then used to calculate the mean fluorescence intensity of a calibration bead in ERF units. These ERF value-assigned beads can be used to calibrate the fluorescence intensity scale of flow cytometers. NIST has assigned ERF values for commercially available sub-micrometer calibration beads with nominal diameters of 100 nm, 200 nm, and 500 nm.
Disclaimer: Certain commercial materials are identified to specify experimental results as completely as possible. In no case does the identification of specific manufacturers, brands, or materials imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials are necessarily the best available for the purpose.
Return to the Quantitative Flow Cytometry Measurements Program page.