How did you prepare your samples for TEM?

We have always used the ImageJ software to calculate the average size of nanoparticles. You can check it out here at https://imagej.nih.gov/ij/download.html

There are instructions you can follow very easily...if your micrograph has a scale bar, you can also manually calculate the average diameter of your nanoparticles with a ruler. Enlarge the picture and then print it out so you can do the measurements.

You can try this software: Nano measurer.

you can try this alternative method to measure nano-particle size using UV spectrometer . It is based on " Mie theory ".

Reham Hamida Please post a micrograph of what you are trying to analyze.

Alan F Rawle Himangshu Dihingia Wenyi Zhou Zoraida P Aguilar Thank you :)

Dear Reham,

As Zoraida says, for this purpose, I have also used ImageJ freeware, together with the freeware Global Lab Image (GLI). Both GLI and ImageJ allow computing, for a given micrograph, a variety of parameters like number of grains, average dimensions, average/min/max grain boundary length, etc.

If you go to the website for downloading ImageJ, the version "ij149 1.6.0_24 (37MB)” works well in Windows XP and Windows 7, Windows 8 and Windows 10.

But first, as Pal mentioned above, you need to make a binary image of your selected boundaries. With ImageJ I usually perform several operations for pre-processing the image to be able to measure better the grain boundary length:

1. Process > Smooth

2. Process > Noise > De-speckle

3. Process > Enhance contrast (at 0.2% or 0.4% without equalizing the histogram)

4. Process > Binary > Make binary (a mandatory step to be able to make the desired computations)

5. Here some editing of the image with Windows Paint/Photoshop/GIMP may be necessary to eliminate imperfections of the image that can distort the computations

6. Process > Binary > Dilate (optional)

7. Edit > Invert (if necessary)

8. Analyze > Analyze particles

Hope this is useful,

Jose

I have the following from Reham Hamida via PM:

Hello, Prof. Alan

thank you for your help

I will attach the photo of TEM micrograph

the method to prepare samples for examination with TEM are as a following

Samples were prepared by drying a drop of the washed NPs dispersion onto the carbon-coated copper grid.

Thank you so much

Reham Hamida I think you have clustered the particles due to evaporation of the continuous phase. This has led to solid bridges between the primary particles which are now aggregates. The transport properties would reflect these aggregates and not the primary size. All your clusters are < 100 nm (so the system is obviously 'nano'). You either need to try and prevent the primary particles clustering. Image J is a route to 'software disperse' the particles but the smaller size does not describe the aggregates you have.

Take a look at this webinar:

May 28th, 2019 Dispersion and nanotechnology

https://www.brainshark.com/malvern/vu?pi=zHSznNgprz5chz0

Also note, that to calculate the mean to 1% standard error requires that you measure 10000 particles and you are a long way short....

Dear professor Alan F Rawle please the meaning of evaporation of the continuous phase related to the drying step ?

Actually i agree with your explanation because during the measurement by using imagej, i couldn't determine the boundaries!

So, your advise to me is "try to prepare other suspension and examine it again using TEM" ?

Thank you

Jose Ignacio Rojas Gregorio We have to be extremely wary of 'software dispersion' as this may not relate at all to the properties of the particles at hand. Take a look at one of the clusters that I will call H1. We could look at this image and (falsely, by eye) conclude that there are 8 particles here. A simple digitization to a 2-color gif indicates 2 particles in total. See H2. Even a rough glance (incorrectly) suggests 7 particles - the issue being shown in H3 where one particle is located above another and probably the solid bridge is hidden from view.. The transport properties of this system reflect the 'larger' sizes. The dangerous step is to software deconvolute the images. I have shown such a deconvolution in H4 where we appear seductively to have 7 particles. While this is an attractive answer it's incorrect as the true nature of the solid bound system is indicated in H2. We could invent some wording such as 'primary size' to help us through his issue but the bulk properties of the system reflect the larger sizes. The surface area could be more indicated by the smaller sizes. It's not as simple as you may think and shouldn't be hidden by a bunch of (software) steps that all look logical and provide the small answer that we're looking for...

I have the following from Reham Hamida by PM:

Dear professor , I'm sorry to write again but after your last comment i became afraid because i'm not specialist in this analysis and it is first time in the field of biological synthesis of NPs and i don't have any supervisor have background about this Science.

So, your comment is supposed that my NPs is larger while in true they may have small range yes or not.

what i must do?

do these aggregation due to the biological material that were used for the production.

thank you

The clustering of the particles is almost certainly due to the sample preparation you used for TEM causing the particles to come together and solid-solid diffusion will do the rest. Please view the webinar.

So the aggregates are an artifact of the sample preparation and almost certainly do not exist in this form in your starting material. However, you have to find a route to stabilize the system (steric or charge) in order to keep the particles separate for correct image analysis. Bear in my my point about 10000 particles minimum. Be wary of 'software solutions' to your issues.

Also, be aware that images are only a 2D representation of a 3D particle.

We have no awareness of the z-dimension.

Thank you prof. Alan F Rawle

Jose Ignacio Rojas Gregorio I really worry about:

5. Here some editing of the image with Windows Paint/Photoshop/GIMP may be necessary to eliminate imperfections of the image that can distort the computations......

Reham Hamida Be aware that there are a bunch of ways of calculating mean values in particle sizing. You need to be familiar with number, length, surface area, volume/mass, and intensity means. See the attached and this webinar:

February 16th, 2017 Basic Principles of Particle Size Analysis

http://tinyurl.com/zo6mfgz

Hi.

use particle size analyzer or TEm we can find it

thanks. it's just that someone told me once that, in the mean time, we are the best processing software.