The Tool Desk
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Start with a representative aliquot, use a clean and compatible diluent, adjust concentration to the validated range of your instrument, and treat every removal step as a potential source of particle loss or aggregation. Record the complete preparation so another analyst can reproduce the same fraction and conditions.
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Why preparation determines the result
Label-free instruments detect particles through their physical signal rather than a fluorescent or chemical tag. That makes them useful for native dispersions, but it also means dust, buffer particles, aggregates, sediment and optical background can be counted or scattered along with the target.
Preparation is therefore part of the measurement, not a preliminary chore. A filter can remove the analyte as well as contamination; centrifugation can deplete large particles; sonication can break weak aggregates or create heat; and letting a sample settle can preferentially remove fast-sedimenting material. If the purpose is to measure the original distribution, each intervention must be justified and reported. If the purpose is to measure a defined fraction, the fraction must be described explicitly.
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ISO/TS 24672:2023 covers guidance for nanoparticle number-concentration measurements across several techniques. Its scope includes DLS, differential centrifugal sedimentation (DCS), multi-detector light scattering (MDLS), small-angle X-ray scattering (SAXS), ultraviolet-visible methods, particle tracking analysis (PTA), resistive-pulse sensing (RPS), single-particle ICP-MS, condensation particle counters and differential mobility analysis systems. These methods do not share one preparation protocol or even measure the same particle property.
Choose the measurement before choosing the preparation
Define the question in operational terms: are you estimating hydrodynamic size, counting visible particles, measuring number concentration, or characterizing another attribute? Confirm that the instrument has been validated for the matrix and particle range before adapting a procedure from another platform.
| Method | What is measured | Preparation priorities | Main interpretation caution |
|---|---|---|---|
| Dynamic light scattering (DLS) | Fluctuations in ensemble light scattering, converted to diffusion and hydrodynamic size | Clean, particle-free diluent; dust-free cell and labware; concentration adjusted for particle scattering; filtration or centrifugation only when compatible | The result is intensity-weighted, so a small amount of large material can dominate the signal and differ greatly from a number-based distribution |
| Nanoparticle tracking analysis (NTA) or PTA | Brownian motion of individual optically visible particles | Dilution to the platform’s usable particle density; diluent blank; assessment of sedimentation and camera settings | Counts depend on visibility, camera, detection threshold and preparation; particles in the diluent can be counted as sample |
| Other number-concentration methods | Technique-specific signals such as centrifugal separation, ionization, electrical pulses or mobility | Use the method’s own validated sample form, calibration and handling instructions | A procedure that works for DLS or NTA cannot be transferred automatically because the measured property and biases differ |
For every method, compare six questions: Is the measurement ensemble-based or particle-by-particle? What output is required? What size and concentration range is validated? How does the matrix affect optical or physical detection? Could preparation remove, dissolve or aggregate particles? Which settings and controls must be reported?
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Take a representative sample
Begin with the sampling plan, not the instrument. Mix or resuspend the source material only as strongly as its stability permits, then withdraw an aliquot that represents the material you intend to report. A protocol for a stable aqueous dispersion may allow gentle inversion; a fragile biological preparation may require a defined number of slow aspirations; a sedimenting suspension may require timed sampling from a specified location.
Do not assume vigorous vortexing, sonication or repeated pipetting is neutral. Those actions can change aggregate size, disrupt vesicles or introduce bubbles. Conversely, insufficient mixing can leave heavy particles at the bottom and produce a falsely low count in the withdrawn aliquot. In regulated food and feed applications, the European Food Safety Authority states that sampling, preparation and analysis should capture the small-particle fraction representatively and that preparation conditions can alter observed size and concentration. The same principle applies to other matrices: define the mixing, waiting time, withdrawal point and aliquot volume in the protocol.
Prepare the diluent and labware
Use a diluent that is chemically compatible with the particle and does not create a background that overwhelms the signal. Match pH, ionic strength, surfactant content and other relevant conditions to the experiment; a change in medium can change aggregation or hydrodynamic size even when the particle itself is unchanged.
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For DLS, the National Cancer Institute Nanotechnology Characterization Laboratory recommends a freshly filtered buffer and dust-free labware. Clean the sample cell or cuvette according to the instrument’s instructions, allow it to dry without lint or residue, and handle it so dust cannot enter. A particle-free blank made from the diluent and the same container is a practical check for contamination.
For NTA/PTA, measure the diluent blank when particles in the medium could be detected. A buffer that looks clear can still contain enough particulate material to affect a low-concentration count. If the blank is not acceptable, identify whether the source is the water, salt, additive, container, filter or handling step before proceeding.
DLS preparation: a controlled sequence
- Confirm the validated sample type. Check the instrument method for compatible solvents, viscosity assumptions, refractive-index inputs, cell type and minimum volume. DLS reports hydrodynamic size inferred from diffusion, so incorrect medium properties can bias the conversion.
- Set a starting concentration. DLS response depends on particle size, concentration, refractive index and optical conditions. Too little material gives an unstable correlation function; too much material can cause multiple scattering or detector saturation. Use the platform’s concentration guidance and plan serial dilutions rather than guessing one final value.
- Mix gently and remove bubbles. Use a defined mixing motion that disperses the sample without creating foam or changing the particles. Load the cell slowly; bubbles and scratches can produce strong artifacts.
- Consider filtration only after checking particle retention. The NCI protocol identifies detergent-free 0.2 micrometre polyethersulfone as one possible low-protein-binding option for many aqueous applications. That is not a universal instruction. Select membrane chemistry and pore size so the principal analyte is not adsorbed, sieved out or otherwise modified. If the target is near the pore size, compare filtered and unfiltered aliquots or use another contamination-control strategy.
- Measure a blank and replicate aliquots. A diluent blank helps distinguish dust or buffer scattering from sample scattering. Replicate preparations reveal whether handling, rather than the particle population, is driving variability.
- Record the complete state. Note dilution factor, diluent composition, filtration details, mixing, equilibration time, cell type, temperature and instrument settings. These details are needed to interpret an intensity-weighted result.
NTA or PTA preparation: dilution, blanks and sedimentation
NTA tracks individual particles, so the suspension must contain enough visible particles for statistics without so many that tracks overlap. The usable density is platform- and setting-specific; camera level, detection threshold, focus and viscosity all matter.
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- Prepare a clean diluent. Filter it only with a membrane known to be compatible with the particles and measure a blank in the same consumable used for the sample. The OSTR Biophysics Core at the National Cancer Institute describes filtered diluent and instrument-specific loading practices for its ZetaView TWIN system.
- Make a dilution series. Start with several dilutions when the stock concentration is unknown. The OSTR facility gives a typical example of a 500- to 1,000-fold stock dilution to reach approximately 107 particles per millilitre on its stated instrument. That figure is facility-specific guidance, not a universal NTA threshold; use the range validated for your platform.
- Inspect for rapidly sedimenting material. Large aggregates or dense particles can leave the optical field during a recording. Decide in advance whether to resuspend immediately before loading, allow a defined settling period, or remove a fraction. Each choice measures a different population and must be reported.
- Load without introducing bubbles. Follow the instrument’s chamber volume and loading sequence. Flush between samples as required, and use the same equilibration and recording times for all replicates.
- Check the blank and quality metrics. A high blank count, unstable focus or excessive coincidence indicates that the preparation or settings need correction before interpreting the sample.
Published NTA work on extracellular-vesicle measurements has found systematic differences associated with preparation choices, diluent contamination and instrument settings. For comparisons across laboratories, harmonize those variables or treat results as method-specific rather than interchangeable.
When to filter, centrifuge, sonicate or let a sample settle
Use a removal or dispersion step only when its effect on the target is understood.
- Filtration: useful for reducing dust or large contaminants when the membrane does not retain, adsorb or alter the target. Report membrane material, pore size, wetting liquid, pressure or syringe technique, and whether the sample was pre-rinsed. Never describe filtration as mandatory for all DLS or NTA samples.
- Centrifugation: can remove visible debris or separate a defined size or density fraction, but it also depletes large target particles and may compact a pellet that is difficult to redisperse. Record rotor type, relative centrifugal force, duration, temperature and which fraction was analyzed.
- Sonication: may disperse reversible agglomerates, yet can fracture particles, disrupt biological vesicles or heat the sample. Specify power, pulse pattern, total time and cooling if it is used. A no-sonication control is valuable when aggregate state is part of the question.
- Settling: allows fast-sedimenting material to leave the measurement zone, but the supernatant no longer represents the original suspension. Define the settling time and sampling height.
- Repeated dilution: changes ionic strength and can shift equilibrium between single particles and aggregates. Use a diluent that preserves the intended chemistry, and verify that the measured distribution is stable across the dilution series.
Matrix and particle-type limits
The title alone does not specify whether the sample is an aqueous nanoparticle dispersion, extracellular vesicles, aerosol, dry powder, food or feed material, or another matrix. It also does not identify particle chemistry, target size, instrument model or whether aggregation is the object of study. Those details determine the acceptable filter, buffer, concentration and handling time.
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NIST maintains separate nanoparticle dispersion preparation protocols, while ISO/TS 24672:2023 addresses multiple measurement methods and sample forms. Use the protocol specific to your matrix and instrument rather than importing a recipe from a different material. For biological samples, preserve the relevant storage and temperature history; for food or feed, document homogenization and the fraction sampled; for dry powders or aerosols, use a validated dispersion or aerosolization procedure instead of assuming that an aqueous dilution is representative.
Interpret the output with the preparation attached
DLS and NTA can both report a particle-size distribution, but the distributions are not equivalent. DLS is dominated by scattering intensity, which gives disproportionate influence to larger scatterers. NTA counts particles that are individually visible and trackable, so low-refractive-index or very small particles may be underrepresented. A concentration or size value is meaningful only with its method, settings and preparation.
When comparing samples, keep the workflow constant: same diluent lot where practical, same dilution sequence, same filtration decision, same equilibration time, same temperature and same instrument settings. If one sample requires a different treatment, treat it as a different method condition and avoid attributing the resulting shift solely to particle composition.
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- Source material, sampling location and mixing or resuspension procedure
- Aliquot volume, dilution factor and final estimated concentration
- Diluent composition, preparation date and blank result
- Filter material, pore size and whether filtration was used
- Centrifugation, settling or sonication conditions, including the fraction analyzed
- Container, cuvette or flow-cell type and cleaning procedure
- Storage temperature, elapsed time and equilibration before measurement
- Instrument model, software version, camera or detector settings, threshold and viscosity or refractive-index inputs
- Number of technical replicates and any excluded recordings, with the reason
This record makes it possible to distinguish a real change in particle population from a change introduced during preparation.
Troubleshooting common preparation failures
| Symptom | Likely preparation cause | Corrective action |
|---|---|---|
| Unstable or very noisy DLS correlation function | Too little sample, dust, bubbles, dirty cell or unsuitable concentration | Run a diluent blank, clean or replace the cell, remove bubbles and test a controlled concentration series |
| DLS size shifts sharply after filtration | Target particles were retained, adsorbed or de-aggregated by the filter | Compare matched filtered and unfiltered aliquots and verify membrane compatibility before using filtration routinely |
| NTA count remains high in the blank | Particles in water, buffer, container or filter | Trace each preparation component, replace the contaminated component and remeasure the blank |
| Large variation between NTA replicates | Sedimentation, inconsistent mixing, changing focus or different time after loading | Standardize resuspension, loading, equilibration and recording times; inspect the field for settling |
| Apparent loss of large particles | Settling or centrifugation removed a fraction before measurement | Measure the original suspension promptly or report the analyzed supernatant or pellet fraction explicitly |
Bottom line for a defensible protocol
Define the measurement objective, sample representatively, use a verified clean diluent and adjust concentration iteratively for the specific instrument. Treat filtration, centrifugation, sonication and settling as experimental variables that can change the measured population. A blank, matched replicates and complete preparation record are more valuable than a supposedly universal dilution or filter specification.
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