Does this technique display a polydispersity index, as DLS does?

NTA tracks and sizes each particle within the field of view on an individual basis. This means that a total particle count at each size is provided and as such the particle size distribution measured is an indication of how polydisperse the sample is. A poly-dispersity index is therefore not required.

What can you tell us about particle shape, eg planar or needle shape

As particles or viruses with a high aspect ratio rotate due to their rotational Brownian motion, they offer different scattering faces which scatter light differently. The aspect with the largest scattering volume will cause the particle to appear bright in the image generated, when the particle is orientated such that the smallest scattering volume scatters light, the particle will appear dim. This results in the particle flashing in the field of view in the NanoSight instrument. As such particles with a high aspect ratio will flash whereas a spherical or icosahedral virus will not flash.  The rate of particle flashing could potentially be linked to aspect ratio. The ability of the NanoSight instrument to image particle flashing is related to the speed of the rotational Brownian motion, which in turn is related to the aspect ratio of the particle.

Can you please tell us what is the range of particle concentrations over which this technique is linear?

The optimum concentration for analysis using the NanoSight system lies between 10^7 to 10^9 particles per ml (remembering that the total virus count provided by NanoSight will be approximately 1-3 orders of magnitude higher than the infective viral titer measured by infectivity assays). Above this concentration, there are too many particles for the software to accurately track. Below this concentration range the user runs the risk of running a non-representative analysis. Repeat experimentation as well as longer analysis times can help improve the statistics when working with low particle concentrations.

Is there a smart way to deal with a high level background solution?

The best way to deal with a high background solution would be through fluorescent labeling of the virus. Viral antigen or potentially viral DNA could be labeled. When operated in fluorescence mode only the labeled virus will be imaged.

The non-specific way of dealing with a high background would be by running a control sample and performing a simple background subtraction.

Since the particles are smaller than the diffraction limit for optical microscopy, how are you able to "visualize" the particles? Is the metalized surface important (e.g., surface plasmons)?

The NanoSight system simply uses light scatter as a way of visualizing and locating particles and viruses within the sample. As these are much smaller than the wavelength incident laser source, then there is no information about the morphology of the particles. The videos that the NanoSight system produces show spots of light that represent particles moving round under Brownian motion. The particles or viruses themselves simply act as point scatterers of light.

The metalized surface of the glass simply gives a the system a good background against which to visualize these particles.

How is it possible to tell virus from cell debris?

In light scatter mode these can only be determined by a difference in particle size. The system can however operate in fluorescence mode and suitably labeled viruses could therefore be specifically distinguished from non-labeled cell debris.

What about biological safety when handling the sample of virus? Can you disinfect the sample holder?

It is possible to dissemble the sample chamber and clean with ethanol, bleach or other cleaning agents that are not likely to etch glass. It is also possible to autoclave the sample chamber, provided the luer fittings and the o-ring have been removed.

Could you distinguish full vs empty particles, of adenovirus for example - likely to be essentially the same size, one with and one without packaged DNA?

Again fluorescence would be the best way of dealing with this. The sample could be measured in light scatter mode which would not differentiate filled or unfilled capsids, as such a total virus count could be achieved.

Then following suitable labeling of the viral DNA/RNA the sample could be measured again, detecting only the labeled viruses. The ability to measured the fluorescently labeled viruses would depend on the quantum efficiency of the fluorophore, binding affinity to the target and life time of the fluorophore.

With regard to gene therapy applications, what virus types have you tested the system upon. We for example largely work on AAV, which may be too small 20-25nm?

Adeno virus and lentivirus have been studied in gene therapy applications. Currently Adeno Assoicated Virus (AAV) is just below the detection limit for the technique however aggregates can be measured and numerated.

Typically, particles much smaller than the wavelength of light do not scatter light efficiently. Why does your instrument work so much more efficiently than any other light microscope?

The optimized optical configuration of the NanoSight instrument is the key to visualizing such small particles and detecting extremely low levels of light scatter.

What viruses have you analyzed and in what media?

The NanoSight instrument has been used to study a large range of virus types in arange of media.

Influenza, adeno, lentivirus, bacculovirus, denge virus, herpes, polio virus are to name but a few. For a full range of sample type please contact a member of the NanoSight team.

You showed us that the Nanosight can measure the number distribution of particles in a solvent. Can the Nanosight also measure the concentration (particles per L) and how does this work?

The NanoSight technique counts all of the particles within the field of view. This field of view has a fixed volume of 120 μm x 80μm x 10μm or 9.6 × 10^-11 litres. The particle count within this fixed volume is then extrapolated to give a particle per ml concentration.

I've heard that an electrophoretic field equipment option is being developed which will allow you to apply a field to the cell and detect electropotential properties. Any update on the status of this capability? Thanks - and great webcast!

This feature is still under development and is currently being beta tested.

Isn't the rotational component too fast for you to measure?

The rotation of the particles is currently not something that the NanoSight system measures.  The size and aspect ratio of the particle will govern its rotational Brownian motion, the speed of which will determine our ability to detect particle flashing with a non-spherical particle.

Is it possible to measure fluorescence of particles?

The latest NanoSight system offers the ability to analyse fluorescent particles within solution. However, the system is only able to monitor the degree to which particles fluoresce, relative to one another, in much the same way the regular system can only monitor the intensity of the light scattered by a particle relative to the others within the sample.

 What sample volume is required?

The sample chamber has a volume of 300μl.

In your fluorescent labeling application would you not also detect labeled antibody that was not bound to a particle?

The NanoSight technique will detect the presence of labeled antibody, not associated with the particle. The labeled antibody, won’t be big enough to resolve on a particle-by particle basis and hence the unbound antibody will cause a slight ‘fog’ of background noise. Providing enough fluorophore is bound to the target particle it will allow the target particle to be individually resolved form the background noise.

Is there any chance that you would come up with a single use cell design? OR alternatively, a CIP (clean in place) friendly cell design?

The latest instrument which are being beta tested at present include a clean in place cell.

Do you have any peer-reviewed scientific publications that demonstrate accurate counting of viruses?

Due to the fact that the majority of interest in this technique for counting virus comes from industry, data is not available for publication. There are many peer-reviewed scientific papers which cite the Nanosight instrument. For a complete list please refer to our website.

How big is the error in size distribution?

As Brownian motion is a random process, even with the most perfectly monodisperse sample, the NanoSight system will detect a slight breadth to the distribution. The longer that a sample is analysed for, the smaller this breadth becomes. To try and negate the random effect of Brownian motion, the NTA Software has a modal curve fit function. After analysis, it is possible to use this feature to try and negate the effect that the randomness of Brownian motion has upon the size distribution.

How do your results correlate with pfu/mL or cfu/mL?

NanoSight counts all particles within the sample, whether they are infective or not. This, along with the fact that plaque assays cannot distinguish between a single infective virion and an aggregate of infective viruses, means that the concentration readings given by NanoSight are often 1-3 orders of magnitude higher than those given by plaque assay.

Does fluoresent labelling techniques help to push the detection limits in terms of particle size lower? Increased signal?

A labeled virus will emit more light than  a non-labeled virus and hence may allow it to be detected at slightly smaller sizes.

Can you detect smaller particles with the lower wavelength lasers?

Lowering the wavelength of the lasers has a minimal effect on the ability for NanoSight to visualize particles within solution, as these particles are still far below the wavelength of the light. The lasers that NanoSight offer that have lower wavelengths

When we recorded the sample at 60, 120 and 180 seconds, we get the particle size at the consistent value. However, the concentration values showed lower at a longer recording time. Do you know what can cause this discrepancy of the concentration?

This could be potentially caused by the virus sticking to the surface of the glass and hence it might be expected that the concentration of particles measured might drop slightly over extended periods of time.

I see you are a British company; do you have USA representation?

Yes, NanoSight has representation in the USA. The contact details are as follows.

USA West Coast Office
Duncan Griffiths
NanoSight USA West
3027 Madeira Ave.
Costa Mesa, CA 92626Tel: 714-747-9955
Email: duncan.griffiths@nanosight.com

USA East Coast Office
Jim Munhall    
NanoSight OH
6660 N. High Street
Suite 2A
Worthington, Ohio 43085Tel: (614) 987-0045
Fax: (614) 888-0025
Cell: (614) 264-3493
Email: jim.munhall@nanosight.com

How much does it all cost?

For further information on pricing please contact NanoSight directly at admin@nanosight.com