Our lab is currently testing the NTA (Nanoparticle Tracking Analysis) technique with the ZetaView from Particle Metrix. However we are really surprised how variable measurements are and how easy it is to get the results you are expecting just by changing the parameters randomly. It seems to have a lot of limitations. Nevertheless NTA is used widely in the EV field.
I tried to measure the fractions of an Optiprep density gradient to get an idea how many particles are present in each fraction. Is it possible to measure all fractions with the same settings and to compare the absolute particle numbers? Particle Metrix told us that different subpopulations might require different settings. That is why I was asking myself how I should adjust the settings to the different EV populations that I find in my gradient fractions.
Thank you very much for your help.
Best, Kathrin
Hello Kathrin,
Although not an expert in the field I do have some knowledge of imaging systems used in industrial settings for particle imaging/tracing and velocimetry.
Regarding your question on the settings to be used on various samples I must presume they are recommended by the equipment manufacturer so as to extract useful data from the images while maximizing sensitivity for a given sample. So the settings to be used take into account camera system performance (pixel size, ISO sensitivity) ,optical system/objective lens performance ( depth of field, f-number, size of Airy disk, etc) and laser wavelength and instrument self-heating . Basically once you have selected a proper wavelength for the particle size and spacing you are expecting to find in the sample the system should be able to measure various particle concentrations but only WITHIN that sensitivity range. Why? Optics. Not only must the individual image of each particle be larger than the average Airy disk of the given system ,but the spacing between two particles that are seen as distinct is also optics-limited. The scatter from two very close objects can sometimes create a single image- that is why when that happens you must change settings to avoid that.
A number of works in the field agree to disagree on "standards" in such measurements because they are all instrument-related . The ISO (sensitivity) performance, the SNR of the sensor, the available laser wavelengths ( limiting factor with respect to measurable particle and space between particle size), the vibration suppression mechanism performance are all deciding factors in both the measurement procedure proper and measurement range/precision. Selecting one or more settings to deal with a gradient of concentrations for example is a matter of whether the system can deal with the maximum concentration in the substance using the same wavelength for laser as for the smallest one- and if no other geometric adjustments are necessary- i.e. no refocus which varies from sample to sample.
All technicalities aside, the core of all imaging methods is getting a good quality..image. That means a good separation of the particles images, no ghosting/flare/unwanted movement traces. That implies a selection of a wavelength, intensity and vibration countering method that can ensure that.Also that means that some settings that would result in the actual image of a particle to be smaller than the average Airy disk /circle of confusion for the given focal length and aperture (are these fixed on your system?) will be avoided and thus sometimes you have to... switch scale just like you do with a regular DMM or oscilloscope- sometimes you sacrifice precision for extended range or you 'zoom in" getting more precision but a limited range- basic measurement theory, but this time- optics limited.
So for the settings- consult the manufacturer/manual and decide if the laser wavelength is tunable (adjustable); if yes, then for each particle /space between particles size you should select one that is comparable to ensure good resolution; if not, that gives you the limit for particle size and spacing thus concentration;for all cases you should be able to select various light levels or various camera sensitivities so that the faintest signal you may get from the smallest detectable particle is still above the noise floor- you can try to find the maximum values for the range of allowable settings in that range, for example.
There are no standards in general in this case because they all relate to measurement system performance, maybe more then they should. Labs choose a measurement procedure in accordance with the stated performances of the instruments and use them as internal standards, it seems.
Many of the settings on your system seem to be automated- the auto mode can thus give you some idea about the system's limitations- in fact , measuring a gradient is a good idea as it shows when the system's limit is reached.
If you have manual overrides on the auto settings (and really such a system should have them) the sensitivity can be increased .
Good luck,
Marius.
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