Dear Yogesh:

Since in a heterogeneous sample, there are "complexes micro/nano-worlds" in each area analyzed, often for comparison, you have to "tune" the sample, in order to optimize the preparation and try to be compatible with both techniques.

The advantage of SEM is that you can start viewing the sample from low magnifications, without having concern for the height of the topography you're watching, which gives you an understanding of where you are and what you are looking at.

For AFM, the situation is much more complex because of the dependence and limitation of maximum area of analysis and maximum Z height imposed by the scanner. For example, the largest size available scanner is approximately 120 microns, this means that you start working on AFM with a magnification of close to 700 times. In these conditions it is difficult to know in which area you are, and especially difficult to position in the optimal or correct area where you know (thanks to other techniques) that there are structures or objects in which you are interested.

In some cases, you can make marks on the reference surface of large dimensions, which help you to position yourself in the area of interest.

For example when we conduct studies of nanoindentation, nano indenter marks are very small (0.5 to 5 microns) separated by distances of tens or hundreds of microns, making it difficult to locate them, to imaging by AFM. In such cases we make much larger in size indenter marks, remarking on the 4 corners of a cube or an imaginary rectangle, the area of interest. But not always is possible to have a system that allows you to make appropriate marks.

Since it is very easy to make mistakes when comparing images from two very different microscopic techniques like SEM and AFM, believing comparing two equal things but they really are not the same things, would have to take certain precautions and procedures to try to ensure that the areas analyzed produce images from the same structures.

So I would recommend first optimize sample preparation as possible, so that the characteristics of the sample are as compatible as possible with the two techniques: SEM and AFM, remembering or keeping in mind that the AFM requires or rather "prefers" samples with low roughness and rather, flat areas and "clean" areas without accumulation of material.

Would try to start with the SEM, analyzing the sample, and selecting those areas more compatible with AFM, and therefore the most promising for good results with AFM.

Would try to seek, macroscopic details, in the sample, such as a torn edge, marks or stains to create useful references in the sample.

In an area of interest and having obtained the images (of the interest structures) with the proper resolution, would attempt to decrease the magnification to match with the magnification of the optical system of the afm microscope, so you have a SEM image with similar size to the image from the afm optical system to help you to position yourself correctly.

In any case it would be necessary to have more information and details to understand the problem you have.

In any case good luck

May be you are getting the variations because of the sample preparation technique,.........which varies quite a lot in both these cases.....

Thanks for quick reply . We have prepared samples with solution growth

What is meant by "different patterns"? If the structures are small enough, the influence of the tip shape in AFM is increasing. However, if your devices are properly calibrated, the pattern should be the same (i.e. the same number of nanoscaled features per area), while the shape of the nanoscaled structures may vary from method to method.

Particle size variation, homogeneity vs heterogeneity of the sample will play important role.

The comparison will be easy if you are recoding the image by non-contact mode in the case of AFM .But if its contact mode you may have to verify many parameter (tip size, area used, environments, etc) to compare the micrograph with AFM Image.

By SEM you detected not only the surface of the sample, because the image formed by thin films of the surface (1-100nm), it dependence with process of the electrons penetration in sample. But quality you can compare result by AFM and SEM. If sample have some features with size 1-5 mkm you can detect this features and by SEM and by AFM. But additional problem fixed this features in area of AFM scan 

In my opinion, to compare images on these scales (micron - nano), because your sample is potentially heterogeneous (depending on what you are looking at obviously), you need to be sure that you are comparing the same area. Experimentally, this might be the most difficult part!

Ideally you should get a comparable image but actually both techniques are based on totally different interactions. Already AFM offers so many different measurement modes that you will get totally different images if you change from mode to mode. The same problem arises if you would ask, why BSE and SE images collected in the same SEM, on the same position, and perhaps simultaneously, look totally different.

Dear Yogesh:

Since in a heterogeneous sample, there are "complexes micro/nano-worlds" in each area analyzed, often for comparison, you have to "tune" the sample, in order to optimize the preparation and try to be compatible with both techniques.

The advantage of SEM is that you can start viewing the sample from low magnifications, without having concern for the height of the topography you're watching, which gives you an understanding of where you are and what you are looking at.

For AFM, the situation is much more complex because of the dependence and limitation of maximum area of analysis and maximum Z height imposed by the scanner. For example, the largest size available scanner is approximately 120 microns, this means that you start working on AFM with a magnification of close to 700 times. In these conditions it is difficult to know in which area you are, and especially difficult to position in the optimal or correct area where you know (thanks to other techniques) that there are structures or objects in which you are interested.

In some cases, you can make marks on the reference surface of large dimensions, which help you to position yourself in the area of interest.

For example when we conduct studies of nanoindentation, nano indenter marks are very small (0.5 to 5 microns) separated by distances of tens or hundreds of microns, making it difficult to locate them, to imaging by AFM. In such cases we make much larger in size indenter marks, remarking on the 4 corners of a cube or an imaginary rectangle, the area of interest. But not always is possible to have a system that allows you to make appropriate marks.

Since it is very easy to make mistakes when comparing images from two very different microscopic techniques like SEM and AFM, believing comparing two equal things but they really are not the same things, would have to take certain precautions and procedures to try to ensure that the areas analyzed produce images from the same structures.

So I would recommend first optimize sample preparation as possible, so that the characteristics of the sample are as compatible as possible with the two techniques: SEM and AFM, remembering or keeping in mind that the AFM requires or rather "prefers" samples with low roughness and rather, flat areas and "clean" areas without accumulation of material.

Would try to start with the SEM, analyzing the sample, and selecting those areas more compatible with AFM, and therefore the most promising for good results with AFM.

Would try to seek, macroscopic details, in the sample, such as a torn edge, marks or stains to create useful references in the sample.

In an area of interest and having obtained the images (of the interest structures) with the proper resolution, would attempt to decrease the magnification to match with the magnification of the optical system of the afm microscope, so you have a SEM image with similar size to the image from the afm optical system to help you to position yourself correctly.

In any case it would be necessary to have more information and details to understand the problem you have.

In any case good luck

Thanks for detailed information

look my friend, you may use the Image Processing toolbox in MATLAB to change the dimension of your SEM micrographs and then you will have a relatively good quantitative comparison instrument. if you are interested, let me no by [email protected]

Atomic force microscopy (AFM) and SEM are two techniques of Scanning Probe Microscopy (SPM) they are useful for imaging of surface structures.AFM is used for High-resolution topographic images of sample surfaces by using a scanning technique for conductors and insulators on atomic scale. A special construction of AFM scanner enables to observe biological samples in liquid environments. Artefacts caused by dehydration of samples are removed this way.. It is strongly sensitive to dehydration and drying that are commonly used in preparation of samples in examinations by Scanning Electron Microscopy (SEM). The two SPM are then complementary methods for better analyses in wet and dry samples

In SEM basic techniques you are getting perspective view on first conductive layer in the material (or on thin layer of gold/carbon deposited to make sample surface from unsuitable material visible in SEM). In AFM you get 3D data representing sample surface shape (convoluted with tip shape for small features) with some artifacts of data acquisition (usually one need to level surface, filter small single-pixel noise on hi-res images, sometimes to make FFT-filtration or remove higher polynomial level features to get good image of surface). If you taking images of conductive sample (or gold-covered) and converting AFM data to 3D view with the same viewing angle and same greyscale pseudocolor, you should get similar images.