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Scanning electron microscopy (SEM) and Bessel beam microscopy are two high-powered microscopy techniques. Whilst Bessel beams can be used in conjunction with SEM instruments, they are both standalone techniques. To understand the key differences, we're focusing on the principles of both these techniques before explaining the main differences between the two.
Scanning Electron Microscopy (SEM)
SEM is a common microscopy technique for scanning a surface. In SEM, a beam of high-energy electrons is fired from an electron gun towards a sample of interest. The beam of electrons is controlled by a series of lenses, scanning coils, deflector plates, and an aperture. As the electrons reach the surface of the sample, they interact with it. When the sample interacts with the incoming electrons, it causes the sample to emit secondary Auger electrons and emit X-rays— both of which are detected. Primary electrons that are backscattered from interacting with the sample are also detected.
When the electrons interact with the sample being imaged, the electrons quickly lose energy due to elastic scattering and absorption interactions, and it is this energy exchange that causes the electrons to reflect off the surface—and does so by elastic scattering. The image is built up by measuring the intensity of the signal produced by the surface. Additionally, the X-rays emitted have specific characteristics that are applicable to each element, so this enables SEM to be used to determine the elemental composition of a surface.
Bessel Beam Microscopy
Bessel beam microscopy is a type of plane illumination microscopy which takes images in a wide field mode and uses different types of structured illumination methods to image a sample. Bessel beam microscopy uses a self-healing beam of light to image a sample laterally in the focus plane. Because these beams are self-healing, any interference that they undergo during the imaging process does not affect the quality of the image, as the beam will re-construct itself after the obstruction.
However, a true Bessel beam is never actually used, as that would require too much energy. Instead, an approximated Bessel beam is generated by firing a Gaussian beam at axisymmetric diffraction gratings or through a lens with a conical surface. A camera is placed at the opposite side of the sample to capture the images. When a sample is being imaged, the sample gets imaged in ‘slices’ (i.e. planes) because the light is fired at a sample in a planar geometry and this enables the microscope to take hundreds of images, which are then stacked on top of each other to generate a 3D picture of the sample.
The Key Differences
The key difference between the two techniques is that an SEM is usually used to image the surface of a material and determine its topological characteristics, whereas a Bessel beam microscope can be used to produce 3D images. Imaging of a surface with SEM leads to a 2D image but showcases spatial variations in a sample. It is possible to construct a 3D image using SEM, but extra methods need to be applied.
The second key difference between the two techniques is in their fundamental operating principles. SEM uses electrons to image the sample by causing them to emit X-rays and secondary Auger electrons, whereas Bessel beam microscopy uses light to laterally scan a sample in three dimensions. Many would think that because light is used in Bessel beam microscopy, that photobleaching and phototoxicity would cause a much lower quality image than SEM, but this is not the case. Because Bessel beam microscopy images in slices, it does not expose the sample to light for extended periods of time, rather it is intermittently. This stops the sample image from becoming photobleached and prevents the sample from phototoxic effects, and in many cases, can lead to a higher quality image than SEM.
There are many other small differences between the two. Given that they both use different mechanisms, the components within the most notable being the detector—an SEM uses a complex electron detector, whereas a camera is simply used in Bessel beam microscopes. The differences also extend to the types of sample that are commonly imaged. Bessel beam microscopes are typically used for smaller materials (especially biomaterials) and are used to image cells and other minute biological matter that are less than 50 microns in size. The main reason being that other planar light-based techniques can not image such small samples. On the other hand, SEM can be used for smaller samples as well as larger surfaces, and larger biological samples, with the average imaging size being anywhere between 5 microns and 1 cm in diameter. One other main difference on the sample front, especially biological samples, is that Bessel beam microscopy can be used on live samples whereas SEM cannot—as the sample needs to be placed in a vacuum to be imaged using SEM. Additionally, whilst both techniques are non-destructive in nature, Bessel beam microscopy is not as energy intensive as SEM.
Sources and Further Reading
- Thermo Fischer Scientific: https://www.thermofisher.com/in/en/home.html
- Carleton College: https://serc.carleton.edu/research_education/geochemsheets/techniques/SEM.html
- NanoScience Instruments: https://www.nanoscience.com/techniques/scanning-electron-microscopy/
- Mapping Ignorance: https://mappingignorance.org/2013/12/23/bessel-beam-plane-illumination-microscopy-another-smart-solution-for-an-old-challenge/
- IMTEK: http://www.imtek.de/professuren/bnp/forschung/Lsmwsrlb
- “3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy”- Gao L., et al, Nature Protocols, 2014, DOI: 10.1038/nprot.2014.087
- “Imaging performance of Bessel beam microscopy”- Snoeyink C., Optics Letters, 2013, DOI: 10.1364/OL.38.002550
- “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination”- Planchon T. A., et al, Nature Methods, 2011, DOI: 10.1038/nmeth.1586
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