The Nanoparticle Characterization Using Sem Biology Essay

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The unwanted part of the image is then cropped. Unwanted parts of an image are those parts, which either are too dark to process, or has multi-layers of the particles. Multi-layers lead to overlap, thereby making it difficult, if not impossible, to distinguish where the particle's boundaries are, and hence, cannot be used to calculate particle size.

Now, the image is converted into an 8-bit format from RGB format so that it can now be converted to binary form either directly or interactively using the 'adjust-threshold' function manually.

If the image contrast and brightness are not good enough for detecting the nanoparticles, it can be adjusted. Also, contrast can be further enhanced using 'enhance contrast' function, if need be.

After all image adjustments, we should have a binary image with particles depicted as black spots on a light/white background. The analysis parameters can now be set to measure the desired parameters. 'Analyze particles' function can then be used to get the areas for all distinct particles.

Sometimes, particles, which touch each other, are detected as one single particle by the software because it fails to find the boundary separating them. These show up with higher than usual areas and have to be removed. Another way to get rid of these is we can set the size range, which is covered during the tracking of the particles.

Also, since the size determined by this method depends a lot on how the threshold was adjusted. So, it is my thinking that the results obtained by this method is not the most reliable measure, even though we are taking an average of numerous measurements.

Figure : (Left) Binary (threshold adjusted) image of the Sample-2. (right) Outlines of the domains found by the software used later to get average area for Sample-2

The average diameter obtained by average 921 particles . This calculation is done under the assumption that the particles are spherical and the cross-section visible in the images is therefore circular.

Question 2

Consider the two size distributions (by intensity and number) determined by DLS. Explain the differences of the obtained size distributions by each analysis


The experimental result that we obtain directly from a DLS experiment is the size distribution by intensity. Number distribution can be obtained using the Mie equation. In typical applications, a number distribution is more desirable since we are generally interested in how many particles of each diameter are present.

The intensity of scattered light is proportional to the square of molecular weight of the materials. It also depends on the size of the particles (I ). An intensity-based distribution can therefore be misleading, for example a small amount of aggregates of larger particles can dominate the distribution. Following were the size distributions by intensity and number for Sample1:

Figure : Size distributions obtained for Sample 1 from DLS

As is generally the case, the mean size obtained from intensity measurements is higher than that from the number measurement. This is because the intensity measurements are biased towards particle sizes, which scatter more light (i.e. towards the bigger particles). Therefore, the whole distribution is shifted towards a slightly higher size.

It should however be noted, that the intensity distribution is what we get directly from the experiments. The number distribution is calculated using the theory developed by Mei (1908). This theory was based on an inherent assumption that there are no errors in the measurement of the intensity data, which is generally not true. Therefore, the number distribution just gives us a better insight in cases where the intensity distribution has anomalous behavior.

Question 3

For one SAXS data set demonstrate how you determined the nanoparticle size


First of all, it is important to make sure we know which solvent was used in the sample under consideration so that we can adjust for the background scattering. In addition, it is also important to know when the sample was taken to determine which calibration curve needs to be used. Calibration is important so that the software understands where the center of the incident beam lies, so that the scattering angles and Q values can be calculated with some confidence.

The first step is to load the calibration file in 'datasqueeze' and center the cross-hair at the approximate center of the bright beam. The hue and contrast are adjusted in the false-color image so that all the rings present in the image are visible. This step, however, has no effect on the actual data, it just adjusts how the false-colors look so that the user does not miss any features during plotting. D=58.38 for the bragg ring used to calibrate the software.

After calibration, the sample and the corresponding background files are loaded. Appropriate multiplying factors are used in order to properly cancel the scattering effects of the solvent background. Once image attributes are adjusted for the clearest possible view, a plot of intensity vs. Q is generated by taking as much of the data as possible in the averaging. Then, an appropriate fitting function is used to get an elementary fit on the experimental data. Parameters are adjusted in order to get the best possible fit, preferably for the 'peaks' at lower Q. The Radius used in fit is the particle radius in Å.

Figure : log (Intensity) vs. Q-value plot for Sample 7

Question 4

The four imaging modes (SE, Z Contrast, BF, HAADF) and the two scattering methods (DLS, SAXS) can be used to determine average size. Even in an ideal situation, the average sizes from the various methods might not be equal. Why?


A very prominent reason why data from scattering methods and from imaging modes are different is the detection limitation. The imaging modes, in general, are less sensitive towards low Z-values. Therefore, elements like C,H,B etc. are not difficult to miss. But this limitation does not exist in case of scattering methods because scattering of light does not depend on Atomic Number. For example, Gold nanoparticles covered with organic surfactants to keep them from agglomeration can be viewed in SEM/STEM, and the organic layer would not be visible. But in DLS, the organic surfactant will act as a coating and make the particle look bigger. Therefore, this will bring a difference in size measured by the two methods.

Another probable cause of this discrepancy may be because the scattering techniques have the particles suspended in a medium where they can freely move, are hydrated, and even possibly under osmotic pressure. These factors do not exist when we are preparing samples for the imaging techniques, they have to be dry, immobilized, covered with a conductive material, and more often than not, under very low pressure. These external factors may have effect on the size/shape of particles, esp in case of soft materials.

Lastly, two different kinds of waves are used in the two techniques. For example, STEM uses an electron beam while DLS uses a laser beam. They have very different wavelengths. If the wavelength of the beam used to study the particles is in order of the size of the particles, then it would affect the measurement accuracy. Since the wavelength of electron beams and laser beams can be quite different, this difference will surface in only one type of measurement, thereby leading to discrepancy in measurement of the same sample by the two methods.

Question 5

Consider all the data available for Sample 1. Report the elements in the nanoparticles and the nanoparticle shape, size, and size dispersity. Comment on your findings


SE gives topographical information. The brightness of the secondary electron signal is proportional to the number of secondary electrons reaching the detector. If the electron beam's angle of incidence is high, the "escape" distance of one side of the beam will decrease, and more secondary electrons will reach the detector leading to bright images. Thus, non-flat surfaces appear brighter than surfaces, which are flat in the SE spectra. From the two SE images obtained for Sample 1, we can observe brightness on circular regions and comparative darkness on other regions hinting to the fact that the particles have a curved surface, i.e. they are probably spherical.

Next we move on to the BS or Z-contrast image for Sample1. Usually, back-scattered electrons give Z-contrast information. This is so because, only those particles, which are made up of high atomic number elements, will be able to efficiently back-scatter or reflect electrons so that they can be detected by a detector placed at roughly 180o with the incident beam. Here, the BSED image is not very conclusive as it is very blurry. This hints to the fact that either the electrons used were not of very high energy because of which they didn't have enough energy to drive them back, or the sample is not composed of very efficient electron reflecting material, i.e. although the sample has a Z-contrast, the highest Z-value is not high enough for electrons to be efficiently back scattered. Later, we find that the highest Z-value is for Fe, which is somewhat expected. We expect to see better BS with elements like Au/Pd etc.

Thirdly, we analyze the Bright Field image. The BF detector is placed along the axis of the incident electron beam and it detects the non-scattered electrons or those in-elastically scattered electrons, which are scattered by a very tiny angle. These electrons have crystallographic information about the sample. In short, the bright areas of BF image are places where the electron beam faced no or little obstruction from the sample. Therefore, bright regions have no sample thickness, or if they do, the Atomic number is low, due to which the scattering is minimal and the beam is able to travel undisturbed. From the Sample1 BF image, we observe dark circular structures showing thick material of comparatively high Z as compared to the periphery of these circular structures. The periphery is light because it is either vacant, or is possibly covered with some organic surfactant which is used to keep the nanoparticles suspended and non-aggregating.

Fourthly, we look at the HAADF image for Sample1. HAADF is done with an annular detector, which captures those electrons, which are scattered after transmission from the sample. As a result, these electrons also contain Z-contrast information because, the those parts of the sample which have higher atomic number elements will scatter almost all the electrons transmitted through them, leading to high reception in the HAADF annular detector. As a result, higher Z-value leads to brighter image in HAADF. From Sample1 HAADF, the previous observation is re-confirmed.

So, till now, we can guess that the nanoparticle is made up of heavier elements as compared to the composition of the surrounding it. Also, it can be reasoned that the particles have a curvature to them, which might mean that they are spherical and not disc-shaped.

EDS data shows peaks for Cu, Fe, Al, C & O. It is hypothesized that these elements are not present in our sample. But they are still showing in our EDS spectra because electrons from the electron gun hit not only the sample but also other parts of the equipment and also the sample stage & holder. These are also composed of atoms, which can release X-Rays because of energetic electrons bombarding their surface. The elements Al, Cu and C are considered to have appeared in our spectra because of this extraneous source and are dropped from analysis. The other peaks are Fe & O. As the cores of the particles are heavier, therefore, we expect a sample with Fe cores and possible coating of an oxide. It is possible to also have a hydrate of the oxide, but since Hydrogen is beyond the capacity of the instrument, there is no way to confirm this.


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging



Question 6

Repeat Question #5 for Sample 2.


SE image: particles appear bright which means they have a curvature to their surface. This means they are probably spherical. Particle aggregates and multi-layers are also bright because they have a non-zero slope which means more electrons deflected towards the SE detector which is at an angle to the incident beam axis.

Z-Contrast: Particles again show up as bright circular spots meaning they are made up of high atomic number elements.

Bright-Field: Particles appear as dark circles with bright periphery. This means they have a heavy core and are either slightly away from each other leaving free space between each other, or have a organic surfactant on the surface, which is common in nanoparticles, to prevent them from aggregating.

HAADF: As expected, particles are bright (near) circular spots showing that particles are indeed made up of a heavy core.

EDS: The material in the core of these particles is expected to be Gold since it is the highest peak. Peaks for Al, Cu & C are neglected because they are considered to be arising out of the experimental set-up.

Table : Size & Dispersion for Sample 2 (for DLS, I have considered only the number averaged diameter since intensity data is skewed to favor the bigger particles)


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging



Remark: In case of SAXS data analysis by datasqueeze software, the dispersion parameter specified is not the actual dispersion in diameter. Rather, D is given as:

Where yi = # photons that hit the ith pixel of the detector and Ei = average of # of photons in 4 surrounding pixels.

Unfortunately, I am not sure what quantity should determine the dispersion in the diameter in SAXS measurement.

Figure : log (Intensity) vs. Q-value plot for Sample 2

Question 7

Repeat Question #5 for Sample 3


SE image: Bright particles meaning curved surface, so probably spherical.

Z-Contrast: Again, this image is not very clear, but has faint impressions of bright circular spots. Therefore, this has two indications- firstly, the particles have a circular cross-section with heavy core and secondly, the composition of the particles probably does not have a third-row transition element, since the back scattering is very faint. (EDS confirms)

Bright-Field: Some dark regions where the layers is so thick that the electrons are unable to penetrate. In other regions, the particles show as dark circular spots because they are expected to have a heavy core, which scatters the electrons and does not allow them to travel along the axis.

HAADF: This image also has some dark spots where probably the layer of particles was so thick that after multiple scattering, the electrons didn't have enough energy to reach the detector. Other regions show bright circular particles with heavy cores, just as other images have suggested and supported.

EDS: Strong peaks of Cadmium and Indium hinting that the cores of the spherical particles are probably made of a mixture of Cd & In. In the core, there is expected to be more Cd and less of In, but this is just a speculation and might not be the case. It depends on how what the Y-axis is on the EDS plot. If it is counts, then Cd>In.


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging



Figure : log (Intensity) vs. Q-value plot for Sample 3

Question 8

Repeat Question #5 for Sample 4.


SE image: Again the particles are similar to the previous samples, but they are not monodisperse. Also, the shape of the particles is not the same and their cross-section is not perfectly circular for most of the particles.

Z-Contrast: The Back-scattering is even fainter than the previous sample indicating that the core of these particles is made up of an even lighter element as compared to the previous samples. (EDS confirms)

Bright-Field: Poly-dispersity is supported in BF image. The particles again show as dark 'near-circular' spots because they are expected to have a comparatively heavier core, which scatters the electrons out of their way, thereby not letting them reach the BF detector along the axis.

HAADF: This image is also as expected from the information obtained from the other data. It shows lesser contrast than the previous HAADF images because the contrast depends on the Z value of the core

EDS: Strong peaks of Silicon and Oxygen. It is difficult to explicitly state whether these particles are Silicon Nanoparticles or Silica Nanoparticles, but they definitely have Silicon in them. This observation supports the shortage of contrast in HAADF image and BSED images.


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging




In this case the data shows strong poly-dispersity. This is verified from the DLS measurement which shows the pDI =0.689. The intensity of the higher diameter is smaller than that of the smaller diameter. In general, a small amount of bigger particle (here 10x bigger) would increase the intensity hugely over the smaller particles. Therefore, this means that the larger particles form a very small portion of the sample and can also be dust particles.

The size distribution by number is found to be very small compared to the size distributions from other measurements. The cumulants fit, however, is in better agreement here with data from other experimental techniques.

Figure : log (Intensity) vs. Q-value plot for Sample 4

Question 9

Repeat Question #5 for Sample 5.


SE image: Particles have a rectangular (or square) cross-section. Their surfaces are expected to be flat and parallel to horizontal in most cases because the brightness is low on the surfaces. In some particles, where the edges are visible, the edges are very bright because of high curvature which leads to more electrons reaching the detector, which itself is at an angle with respect to the incident beam.

Z-Contrast: The Back-scattering has much better contrast and features than the previous sample with Silica/Silicon cores. This means that the sample has a heavier core than Silicon.

Bright-Field: BF images confirm the shape of the particle as one with rectangular cross-section. In addition, multiple layers of particles can also be seen.

HAADF: This image is also as expected from the information obtained from the other data. It shows rectangular particles with bright cores and dark periphery.

EDS: Strong peaks of Iron and Oxygen. In addition we also see a peak for Zinc. It is therefore possible, that the nanoparticles are of an alloy of iron and zinc. Zinc is often added to iron as a sacrificial component to prevent oxidation of Iron because it is more electropositive.

Figure : log (Intensity) vs. Q-value plot for Sample 5


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging




The particles here are not spherical, but are rather expected to be cuboidal (or cubical) Therefore the size measured here in nm by DLS is the hydrodynamic radius of the particle. Because the fit is very poor, the data from DLS is not considered to be reliable. Also these particles are found to be much bigger in size by DLS. Another reason for such a discrepancy could be the solvation of the nanoparticles by the solvent. This might lead to the apparent size of particles to be higher than what they actually are because the size is measured by light scattering and it cannot distinguish whether the aggregate is one particle or a particle surrounded by a mesh of polymers/surfactants/solvent molecules, etc.

This effect is not visible in the other two techniques, which cannot 'see' the surfactant surroundings.

Size obtained from SAXS is also not very reliable because the function used to fit the data was Rayleigh, which is not for cuboidal particles. Still, the radius obtained from this method is found to be in close agreement with the data obtained from STEM BF image.

The STEM BF image was analyzed using imageJ as described in Question1. The area calculated was then equated to get the length of a side of the square, which the cross-sectional surface of the particles have been assumed to be.

The most reliable information for Sample5 should therefore be the information obtained from STEM BF using imageJ, even though it has the uncertainty due to threshold technique, also previously mentioned.

Question 10

Repeat Question #5 for Sample 6.


SE image: Particles have a rectangular (not square) cross-section. Their surface is not expected to be flat because of the high brightness on the surface. Therefore, they are not just cuboidal particles. In addition, in the close-up view of the SE image, we can see contrast lines running parallel to the longer edge of the particles. This pattern is consistent with non-circular cross-section. Therefore, it is expected that the particle shape is probably a rectangular prism with a cross section of either a pentagon, or a hexagon, or something similar.

Z-Contrast: The Back-scattering has much better contrast and features than the previous samples with Fe/Zn and much better than one with Silica/Silicon cores. This means that the sample has a heavier core made up of probably a third row transition metal, which is found to be gold by EDS.

Bright-Field: BF images show that the shape of the particle is one with rectangular cross-section, most probably a rectangular prism. In addition, some particles have smaller cross-section area, which is probably because they are standing on their base.

HAADF: This image is also as expected from the information obtained from the other data. It shows rectangular particles with bright cores and very strong contrast hinting towards presence of Gold, which is then confirmed with EDS.

EDS: Strong peaks of Gold suggests that these nanoparticles Gold nano-rectangular-prisms. There isn't enough evidence to comment on the cross-sections shape.

Figure : log (Intensity) vs. Q-value plot for Sample 6


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging




Dynamic Light Scattering data is again giving us the hydrodynamic radius, which is the radius of hypothetical hard sphere that diffuses with the same speed as the particle under consideration. Since the particle is not spherical, this radius is not of much practical importance, even though it is a good measure of how good the particle diffuses.

From the SAXS data is not the most accurate we can get because the cylindrical function was used for particles, which are actually rectangular prisms. But this is not a very bad approximation. Still, the fit is not very good because the chi square value for this fit is roughly 67.6, which means that the fit is not statistically sound even though it may look that it fits. It might be missing some important statistical parameters necessary for fitting. This is possible if the error bars have been underestimated.

From the STEM data, the area for the rectangular cross sections is obtained. Then the aspect ratio of the particles was taken to be roughly 2 (by observation). Then the area of the rectangle was equated with the empirical area to get the length of the longer edge and the diameter of the cross-section. The value in table is the diameter of the cross-section.

Question 11

Repeat Question #5 for Sample 7. Note that Sample 7 is a mixture of two of the other samples. Identify which two samples have been mixed.


SE image: We can see particles with bright surfaces meaning they are not flat. Since they have a circular cross-section it is safe to assume that they are spherical particles.

Z-Contrast: The Z-contrast is poor and if we look closely, two different kinds of Z-contrast, not very different from each other can be observed. It is therefore possible that two different materials are present in the sample.

Bright-Field: Two different particle-types can be clearly observed in the BF image. They appear distinct because a BF image relies on particles not being able to scatter the e-beam. But if we have a mixture of Z-values, the ones with higher Z will appear darker since most of the electrons entering them have been scattered.

HAADF: A similar thing is observed in the HAADF image where the Z-contrast between the two particles of different Z-values can be clearly seen. Here, the same particles that were dark in the BF (high Z) are brighter. This is because whatever electrons were not reaching the BF detector was going to the annular HAADF detector.

EDS: Peaks suggest presence of Cadmium, Indium, and Iron. Therefore, the heavier element is Cd-In mixture (2nd Transition Row) and the lighter one is Fe (1st Transition Row). Also, This helps in concluding that the sample 7 is a mixture of Samples 1 & 3.

Figure :

Figure : log (Intensity) vs. Q-value plot for Sample 7


Size (nm)

Dispersion (nm)

Dynamic Light Scattering (by #)



Small-Angle X-Ray Scattering



STEM- Bright Field Imaging




The average size of the particles from the three measurement techniques is close. The standard deviation in DLS data is very high as compared to the previous values of the same variable. A possible reason for this could be that one of the particles (1 or 3) can have organic surfactants on their surface. These make the particle appear bigger in DLS but these organic layers cannot be detected by STEM or the SAXS because they cannot resolve at this low Z. Therefore, it is possible that one of the particles appear bigger but only in DLS and not in STEM, leading to high dispersion only in DLS.

Question 12

What are two advantages and two limitations of each method: SE, Z Contrast, BF, HAADF, EDS, DLS, and SAXS for particle characterization? You may compile your answer in a table.





Secondary Electron Detection

Image resolution of up to 0.5nm

Information about Topography even for thick samples

Sample must be conductive otherwise charging

Cannot measure in-situ or inside a medium. Must be immobilized


Intensity ~ Z1.7-2 & th1, so easy to interpret contrast images

Dynamic focus corrects for tilt-induced defects

TEM can be done more quickly, although with lesser resolution

Bright Field

Very simple to set-up and use

Lower electron dose and better control on brightness than TEM

Small depth-of-view, so easy to go out of focus

Thickness limitation


Chemical Analysis along with imaging

Quick collection of full spectrum

Limitation to thickness of features

Sensitive to noise


Quick Measurements

Averaging over thousand of particles

Doesn't need them immobilized or conductive

Less characteristic X-Rays at higher voltage

Low count rate and poor energy resolution as compared to WDS

Dynamic Light Scattering

Practically no limit on size measurable

Sample doesn't have to be a crystal i.e. easy & broad range of samples

Sensitivity to vibrations esp. for small particles

Sensitivity to Temperature


No special sample preparation required before analysis

Can be used in high-throughput experiments

Takes a few minutes

Spatial Averaging leads to loss of available information

Atomic Number below Boron are undetectable