- 1 서문 (Foreword)
- 2 적용범위 (Scope)
- 3 배경 (Background)
- 4 절차 (Procedure)
- 4.1 시료 준비 (Sample preparation)
- 4.2 측정 (Measurement)
- 4.3 Image pre-processing
- 4.4 Analysis
- 4.5 데이터 분석 (Data analysis)
- 4.6 보고서 작성 (Report)
- 5 사례 연구(Case study)
- 6 참고문헌 (References)
Nanoparticle tracking analysis (NTA)
This protocol specifies a method for the application of nanoparticle tracking analysis (NTA) to the estimation of an average hydrodynamic size and measurement of the broadness of the size distribution of mainly submicrometre-size particles dispersed in liquids.
유체역학적 크기(Hydrodynamic size)
The hydrodynamic size is defined as “the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured”. In practice though, particles or macromolecules in solution are non-spherical, dynamic(tumbling), and solvated. Because of this, the diameter calculated from the diffusional properties of the particle will be indicative of the apparent size of the dynamic hydrated/solvated particle. Hence the terminology, Hydrodynamic diameter. The hydrodynamic diameter, or Stokes diameter, therefore is that of a sphere that has the same translational diffusion coefficient as the particle being measured, assuming a hydration layer surrounding the particle or molecule.
NTA (Nanoparticle Tracking Analysis)
Nanoparticle tracking analysis (NTA) is a method for visualizing and analyzing particles in liquids that relates the rate of Brownian motion to particle size. The rate of movement is related only to the viscosity and temperature of the liquid; it is not influenced by particle density or refractive index. NTA allows the determination of a size distribution profile of small particles with a diameter of approximately 10-1000 nanometers (nm) in liquid suspension.
Particles suspended in a fluid show random moving resulted from the bombardment of fast-moving solvent molecules, i.e. Brownian motion. The following equation refers to the relationship between mean squared displacement (MSD) and diffusion coefficient of particle showing Brownian motion for a certain period of a time.
<Δx2> = 2nDτ---------- (1)
where Δx is the displacement of the Brownian motion of the particle over a period of a time, τ, brackets <∙> refer to the statistical average of the contained quantity, n is the number of a translational degrees of freedom, and D is the diffusion coefficient. In the two dimensional Brownian motion, n is 2, so Eq. (1) becomes <Δx2>=4Dτ. The Eq. (1) can also be changed to following equation having more various variables through the Stokes-Einstein equation.
<Δx2> = 2nτ(kBT/6πηr3)---------- (2)
where kB is the Boltzmann’s constant, T is the absolute temperature, η is the viscosity of medium, and r is the hydrodynamic radius of particle. In this experiment, displacements of the specific lag time were gained from the trajectory of each particle showing Brownian motion in the two dimensional plane. When the relation curve of MSD and specific lag time was plotted on a graph, the value of slope of curve was converted to the diffusion coefficient through the Eq. (1) and to the viscosity of medium or the hydrodynamic radius of particle through the Eq. (2).
The technique calculates particle size on a particle-by particle basis, overcoming inherent weaknesses in ensemble techniques such as dynamic light scattering. Since video clips form the basis of the analysis, accurate characterization of real time events such as aggregation and dissolution is possible. Samples require minimal preparation, minimizing the time required to process each sample.
NTA currently operates for particles from about 10 to 1000 nm in diameter, depending on particle type. Analysis of particles at the lowest end of this range is possible only for particles composed of materials with a high refractive index, such gold and silver. The upper size limit is restricted by the limited Brownian motion of large particles; because a large particle moves very slowly, accuracy is diminished. The viscosity of the solvent also influences the movement of particles, and it, too, plays a part in determining the upper size limit for a specific system.
The technique is used in conjunction with an ultramicroscope and a laser illumination unit that together allow small particles in liquid suspension to be visualized moving under Brownian motion. A laser beam is passed through the sample chamber and the particles in suspension in the path of the beam scatter light in such a manner that they can be easily visualized via a long working distance, x20 magnification microscope onto which is mounted a video camera. The camera captures a video file of the particles moving under Brownian motion. Computer software is then used to track the motion of each particle from frame to frame. The rate of particle movement is related to a sphere equivalent hydrodynamic radius as calculated through the Stokes–Einstein equation.
시료 준비 (Sample preparation)
The NTA is configured to operate optimally in aqueous-based samples. For routine analysis of samples in which particles are suspended in non-aqueous solvents, it may be necessary for the manufacturer to adjust the device to accommodate solvents of refractive indices outside the range 1.30 – 1.36. While the device can be used to visualize particles in any solvent type, the suspending medium must be sufficiently non-absorbing, non-index matching and gas free.
a) Transparent Solvent
For the particles to be visible, it is necessary for the solvent in which they are present to be sufficiently transparent to the illumination beam.
b) Non index matching
Similarly, the refractive index (Ri) of the solvent must be sufficiently different from that of the particles otherwise they will be effectively index matched and therefore invisible. The light scattered by the particle (Is) varies as a function of the square of
the refractive index ratio (d) between the solvent and the particle. It is clear therefore, that high refractive index particles (inorganics, metals etc.) present in low Ri solvents will be more visible (i.e. detectable at smaller sizes) than weakly scattering
systems such as low molecular weight biological macromolecules.
c) Formation of bubbles
The device contains a high-powered laser and can get warm over extended periods of use. This may cause any dissolved gasses in a sample to produce bubbles which may degrade image quality. In such cases it is recommended that the sample be degassed prior to use.
The sample must be diluted to a number concentration of between 106 and 109 particles per millilitre (depending on particle type) and not contain particles larger than 10 μm diameter. Such particles will degrade image quality and may sediment in the sample chamber necessitating frequent cleaning of the optical surfaces.
Particle size range applicable
The NTA allows particles as small as 10-20 nm to be visualized (depending on particle and solvent type) and will allow particles as large as a micron to be sized. However, large particles (>1 μm) will scatter significant amounts of light and may mask the presence of smaller particles if present at too high a concentration. Similarly, very large particle aggregates (>10 μm) may affect image quality or might block the sample inlet/outlet ports and should be removed by filtration or centrifugation before the sample is analysed.
For particles to be resolved on an individual basis, it is necessary for the sample to be diluted to a particle number concentration of <109 per ml though this limit will vary with sample type. It is best to adjust sample concentration until a clear image is obtained of a population of at least 100 particles in the scattering volume. The laser beam is focussed, on manufacture, to generate a beam waist of approx 100 μm width of which a length of beam of between 100 μm is observed.
- Switch off the laser and disconnect the power wire.
- Empty the sample chamber by pushing out all residual fluid by syringe via the Luer fittings.
- Use the tool on the LM10 unit power wire to disassemble the screws securing the top plate in opposite diagonal pairs to ensure balance, and lift off the top plate.
- Clean the inside of the window by gently wiping it with an optical-grade lens-cleaning tissue wet with ethanol, acetone or DI water.
- An air stream from a tank of compressed air should be used to blow any residual liquid from the Luer fittings.
- Carefully remove the glass optical flat in the upper surface of the device by inverting the unit so the optical flat drops out onto our hands.
- Wipe the surface of this element with a tissue wet with ethanol, acetone or DI water. Take care not to scratch the optical flat surface, or introduce particulates or contaminants onto the surface.
- When all optical surfaces have cleaned and dried, lay them back into the unit. Reassemble the screws on the plate in opposite diagonal pairs, do not overtighten.
The LM10 unit is now ready to inject a sample.
Load the sample into the sample chamber using a syringe (without needle). The Luer fittings are designed to accept standard syringe bodies of all sizes. Avoid the introduction of bubbles at this stage. Bubbles in the sample will degrade image quality because they are very effective light scattering centers. The bubbles must be removed before viewing through the microscope
The sample should be injected slowly and vertically upwards. The maximum sample volume that can be injected into the chamber is 0.5 mL. A properly prepared and loaded sample will appear as a clear sample through which the laser beam can be seen as a thin line passing through the sample chamber. If the beam appears very bright and appears to ‘bloom’ within the sample, the sample is too concentrated and must be further diluted before viewing through the microscope.
To remove a sample, simply extract it using a syringe. The chamber can be cleaned by flushing through the chamber using DI water before loading a different sample. A tissue should be held at the output port to soak up the excess water from flushing. Between samples which have different solvent or particle type, it is necessary to disassemble the top plate and rinse and dry thoroughly the optical window and flat as described above.
Once the sample is injected, place the LM unit onto the microscope stage and adjust the position and height of the microscope objective to obtain a clear image of particles present within the beam. Make sure the tip of the objective does not touch the window of the LM10 unit, because that may break or crack the optical window. Use the microscope stage X and Y adjusters to locate the portion of the beam traversing the chamber which gives the best image.
1. NTA Software usage
Click on the NTA icon to open the program. 
On opening the NTA program, the following screen appears.
The user has 2 choices:
- Capture a video from the camera for saving or immediate analysis.
- Open File to load a previously captured video for (re-)analysis.
Depending on choice, the program will either.
- Choice1 - Capture a video →Go to the Capture Screen
- Choice2 - Open File → Load a *.avi video file (where * denotes the file name chosen when captured and saved.
2. Video capture
The video capture interface contains the camera control interface and live display.
In the camera control interface is responsible for setting camera controls manually. (standard and advanced)
- Standard mode is enough for most samples
- Advanced mode is only used for when the user need better precision in the camera settings or recording duration.
3. Camera Level – Determines the brightness of the image.
To check camera level in the image stream or When the triangle is shown up on screen, the user should be control the camera level.
The 'BRIGHT!' warning indicates that a high amount of saturation has been detected in the image stream.
The camera level should be reduced unless this causes the dimmest particles to disappear ; in which case it may help to reduce concentration slightly
The 'DARK!' warning indicated that the image has a low light level, which can cause the analysis to only track the brightest particles in the image.
The camera level should be increased to ensure that all particles in the samples are being visualized.
4. Record – Starts the video capture.
5. Input temperature – At the end of the video capture sequence, the user is required to input the temperature of the sample. The temperature can be obtained with the temperature probe that is supplied with the instrument.
6. Save file – After entering the temperature, the user is required to input a file name for the video to be saved and a location where it can be saved. The video is saved with the default .AVI extension.
7. Capture duration – Determines the length of the video. Suggested capture duration is selected from a table based on approximate concentration (particles per image) and polydispersity.
Screen Gain – Adjust the brightness of the image displayed on screen with no effect on detection. Use this setting to help visualize the dimmest particles when setting the detection threshold.
Detection Threshold – determines the minimum grey scale value of any particle image necessary for it to qualify as a particle to be tracked for analysis. When a particle has been correctly identified as a trackable object, a red cross will appear at the particle center.
- Too high threshold may result in loss of particles from the analysis.
- Too low threshold may result in noise being incorrectly identified as particles.
Temperature & Viscosity – The temperature and viscosity of the solvent needs to be set before processing and analysis begins. These values are automatically input when a video is captured.
By checking the tick box next to the Temperature slide bar, the temperature will automatically be linked to the viscosity of water. For samples not dispersed in water, uncheck the box and enter the viscosity value for the solution at the indicated temperature.
Process sequence – Starts the analysis of the captured video sequence based on the preprocessing settings.
The result will be displayed in 3 plots: Particle Size Distribution plot, 2D Intensity vs. Size scatter plot and 3D Intensity vs. Size vs. Number plot.
In addition, the software will export a .CSV file, which contains the particle size counts, so that the users can reproduce and modify the Particles Size Distribution plot by themselves.
데이터 분석 (Data analysis)
보고서 작성 (Report)
시험보고서에는 다음과 같은 사항들이 기재되어야 한다.
c) 시료 정보 : 시료명, 제품번호 등
d) 시료의 분산 용매
e) 시료의 전처리 방법
h) 측정된 입자의 유체역학적크기 및 크기분포의 평균값 및 표준편차
i) 측정 및 분석시 시험에 영향을 준 비정상적 특징