SEM is a very useful technique for analysis of bone in association with implanted materials.27–29 The technique is also valuable for the identification of relevant areas which may be selected for further analysis, using, for example, the new focused ion beam microscopy (FIB) technique.
From: Bone Repair Biomaterials, 2009
Related terms:
T.D. Allen, M.W. Goldberg, in Encyclopedia of Cell Biology, 2016
SEM of Subcellular Detail
SEM of internal cell structure may appear nonintuitive, given the familiarity of SEM images of whole cells and tissues viewed from the surface, but separation of cell contents (largely for biochemical analyses) has been an integral part of cell biology since its inception. Isolated organelles such as mitochondria or other vesicular components can be purified and visualized by SEM, or even a molecular level of purification for cytoskeletal elements such as tubulin, which can then be repolymerized in vitro to form intact microtubules and viewed by SEM. The resolution limits of FEG-SEM were pushed in the early 1990s by Martin Müller and colleagues, who produced stunning images of bacteriophage substructure, DNA, Fab fragments, and ultra-small immuno-gold particles (Hermann et al., 1991). One of the earliest candidates for the enhanced 3D imaging provided by SEM was the chromosome, a robust structure due to the packaging ratio of 10 000:1 for the length of chromosome compared to the length of DNA contained within it. Whole chromosomes proved fairly impenetrable to early TEM studies producing a dense silhouette with a few individual looped fibers at the periphery. SEM of chromosomes that had been originally prepared for human karyotype analysis showed the whole structure in good surface detail. SEM imaging also revealed structural alterations along the length of the chromosome when LM staining protocols such as Geimsa were used to produce banding patterns to aid chromosome analysis. In the case of the Fragile X syndrome (which causes learning disabilities and cognitive impairment), the potential break point was precisely located by SEM studies (Harrison et al., 1983). As with whole cell images, SEM images of chromosomes have proved popular for the majority of textbooks (Figure 5).
Figure 5. Chromosomes from a dividing muntjac cell in which the DNA was experimentally altered during replication, resulting in a structural difference between the chromatids which is clearly visible in SEM
(courtesy C.J. Harrison).
Y. Homma, in Encyclopedia of Materials: Science and Technology, 2001
4 Summary
SEM allows the observation of surface morphology evolution due to two-dimensional island nucleation and coalescence in GaAs MBE and MEE. SEM images reveal various stages of layer-by-layer growth. The step propagation velocity can be analyzed from the images. The advantage of in situ SEM is that it can image surfaces without growth interruption in a wide range. The vertical resolution is high enough to detect an atomic layer, while the lateral resolution is 2–5 nm, depending on the working distance and acceleration voltage. The contrast is not very high, so rather slow scanning is necessary.
Anne L. van de Ven, … Rita E. Serda, in Methods in Enzymology, 2012
3.2 Loading characterization
Scanning electron microscopy (SEM) can be used to characterize LEVs after loading. This technique uses a narrow electron beam to collect high-resolution, high-magnification images of backscattered electrons emitted from sample surfaces. Due to the narrowness of the excitation beam, the resultant images have a high depth-of-field that can be used to understand particle topography. Since an SEM of sufficient resolution can distinguish individual NPs within the pores of pSi particles, we use SEM on a regular basis to confirm pSi loading, evaluate the nature of NP loading, and qualitatively determine loading efficiency. Sample preparation is simple and requires only a small amount of LEVs (∼ 106) to be dried on a SEM stub and generally uses much less sample than destructive, time-consuming, chemical-based assays.
Vandana Patravale, … Ratnesh Jain, in Nanoparticulate Drug Delivery, 2012
Scanning Electron Microscopy (SEM) [4, 12]
SEM is an ideal technique to assess the purity, extent of aggregation and degree of dispersion and homogeneity of nanoparticles. The main advantage of this technique is the prevention of sample destruction in Environmental- or E-SEM mode; the measurements correlate with relative humidity of real atmospheric conditions by suitable variations in the vacuum and temperature inside the sample chamber [36]. Thus the technique is advantageous especially for polymeric nanoparticles since the morphology can be visualized in liquid state since complete drying of polymeric nanoparticles may alter their inherent morphological characteristics.
However, the application of SEM is limited since it sometimes fails to distinguish between the nanoparticles and the substrate. The inadequacy of its resolution becomes even more pronounced in systems tending to form agglomerates in which case TEM provides a more feasible alternative to minutely visualize the structural nuances of such nanoparticle clumps. Moreover, in E-SEM the movement of particles in liquid film and lack of conductive coating leads to compromise in the resolution of the final images [36]. Also as with TEM, the sample size is statistically small to reveal an absolute representation of the bulk nanoparticles.
A representative SEM image of polymeric nanoparticles of docetaxel formulated using a hydrophobic starch polymer has been depicted in Figure 3.4(a). Imaging was performed after sputter-coating of the sample with gold, under vacuum, to enhance the contrast. Figure 3.4(b) depicts the E-SEM image of the same nanoparticle system. As described earlier the ESEM image exhibits a lower contrast as compared to SEM. However, the images comply with each other in terms of average particle size and sample homogeneity. Also no bridging of the nanoparticles is observed in either case.
Figure 3.4. (a) SEM and (b) E-SEM image of polymeric nanoparticles of docetaxel formulated using a hydrophobic starch polymer. The SEM image was captured after sputtering the sample with gold to facilitate enhanced resolution.
(Reproduced with permission from John Wiley and Sons [36])
A brief description of some SEM instruments which can find industrial application for the analysis of drug delivery nanoparticles has been presented below.
Ashok K. Singh PhD, in Engineered Nanoparticles, 2016
4.9 Scanning Electron Microscopy
SEM is widely used to investigate the microstructure and chemistry of a range of materials. The main components of the SEM include a source of electrons, electromagnetic lenses to focus electrons, electron detectors, sample chambers, computers, and displays to view the images (Figure 17). Electrons, produced at the top of the column, are accelerated downwards where they passed through a combination of lenses and apertures to produce a fine beam of electrons. The electron beam hits the surface of the sample mounted on a movable stage under vacuum. The sample surface is scanned by moving the electron-beam coils. This beam scanning enables information about a defined area of the sample. The interaction of the electron beam with the sample generates a number of signals, which can then be detected by appropriate detectors.
Figure 17. Components of scanning electron microscopy (SEM).
SEM consists of an electron source that fires a beam of electrons at the object under examination. The beam goes through a couple of lenses made of magnets capable of bending the path of electrons. By doing so, the lenses focus and control the electron beam, ensuring that the electrons focus onto the specimen. The sample chamber is sturdy and insulated from vibration. The sample chambers also manipulate the specimen, placing it at different angles and moving it in all directions. SEM contains various types of detectors capable of (1) producing the most detailed images of an object’s surface and (2) revealing the composition of a substance. The key point is that SEMs require a vacuum to operate. Without a vacuum, the scattered electron beam would distort the surface of the specimen.
The advantages of SEM include the detailed three-dimensional (3D) topographical imaging and the versatile information obtained from different detectors. The microscope is easy to operate and associated software is user-friendly. The disadvantages of SEM are its size and cost. SEM is expensive to operate. The preparation of samples can result in artifacts. A critical disadvantage is that SEM is limited to solid, inorganic samples small enough to fit inside a vacuum chamber that can handle moderate vacuum pressure.
The following manuscripts describe the application of SEM to decipher the surface properties of nanoparticles: de Siqueira et al. (2014), Polyakov et al. (2014), Brown and Hondow (2013), Ponce et al. (2012), Hatziantoniou et al. (2007), Masotti et al. (2007), and Puchalski et al. (2007).
Aaron Elbourne, … Russell J. Crawford, in Methods in Microbiology, 2019
2.1.2 Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) has been a standard technique for the visualisation of biofilms for many decades. A standard model for bacterial biofilms is one formed using Bacillus subtilis. This species can form highly structured colony biofilms and as such, is one of the most studied Gram-positive model microorganisms. These biofilms have been used to help decipher genetic regulation. Typically, to visualise biofilms using SEM, they are grown on a substrate (or surface), chemically fixed, dehydrated, freeze fractured, critical point dried, then sputter coated and analysed. Emerging methods in SEM using a focused ion beam (FIB) have been able to demonstrate important cross-sectional information about the structure of biofilms. The technique, while commonly used, has many disadvantages; these include the formation of destroyed or amorphous layers within a sample, and the fact that it is a very time consuming technique. Ideally, FIB-SEM should be used in conjunction with other techniques to allow comprehensive information about a biofilm to be obtained.
G.E. Amidon, … D.M. Mudie, in Developing Solid Oral Dosage Forms (Second Edition), 2017
10.2.2 Scanning Electron Microscopy
Scanning electron microscopy (SEM) is another technique where only milligram quantities of material may be used to determine particle size, shape, and texture. In SEM, a fine beam of electrons scan across the prepared sample in a series of parallel tracks. The electrons interact with the sample, and produce different signals which can be detected and displayed on the screen of a cathode ray tube (Retrieved from http://www.authorstream.com/Presentation/naveen.gokanapudi-1764246-preformulatio).2 Particles less than 1 nm can be viewed and, since the depth of focus is so much greater than that of the light microscope, information on surface texture can be generated. SEM requires more time-consuming sample preparation than optical microscopy and cannot distinguish between crystalline and noncrystalline materials. It is also more difficult to generate a PSD using SEM since, while the information obtained is visual and descriptive, it is usually not quantitative since only a few particles are seen in the viewing field at one time. However, when SEM is used with other techniques (Retrieved from http://www.authorstream.com/Presentation/naveen.gokanapudi-1764246-preformulatio), it can provide additional valuable information that may help to explain powder properties such as agglomeration or flow problems.
J. Webb, J.H. Holgate, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003
Image Analysis
SEM produces electronic/digitized images and as such they are available for computer manipulation, for example, in image processing or analysis. Image processing involves computer filtration and enhancements to improve the final image quality from the microscope and this type of facility is often a standard feature on modern instruments. Image analysis is used to obtain quantitative data directly from the images, for example, relating to size and size distribution and shape of features as well as relative areas and coincidence measurements. Many purpose-built image analyzers are available commercially with which analysis of any image source, e.g., objects, photographs, light microscopy, and TEM images, as well as SEM images, can be undertaken. The basis for discrimination of features/areas of black and white images, such as SEM images, is the difference in gray levels (differences in black to white) across the image. SEM images often cause problems because of the very wide range of gray levels within any one image, although use of high-contrasting techniques, in particular backscattered imaging, can help eliminate the problem.
Maryam Khan, … Shamoon Asmat, in Methods in Enzymology, 2018
3.7.2.3 Scanning Electron Microscopy and Energy Dispersive X-Ray Spectroscopy (EDS)
SEM studies were performed to analyze the surface morphology of the synthesized NC before and after conjugation with lipase while EDS was employed to analyze the elemental composition of synthesized NC.
Methodology1.
Prepare aqueous samples of ANL and PANI/Ag/GONC bound ANL and ultrasonicate them for even distribution.
2.
Using a micropipette drop-cast 10 μL of the sample on a glass cover slip and blot excess solution using filter paper.
3.
Use JSM SEM instrument to record images.
4.
Using INCAx sight EDAX spectrometer, elemental composition of NC was analyzed.
Monique Y. Rennie, … S. Lee Adamson, in The Guide to Investigation of Mouse Pregnancy, 2014
Chapter Summary
Scanning electron microscopy (SEM) and three-dimensional (3D) micro-computed tomography (micro-CT) are high-resolution imaging techniques that can be used to visualize the structure of the uteroplacental and fetoplacental circulations. In both cases, a cast of the 3D circulatory structure is made by perfusion of a liquid agent through the circulatory bed, which solidifies prior to imaging. SEM images of casts with tissue removed reveal the topology of all vessels, including the smallest vessels in the microcirculation. X-ray imaging of casts with tissue intact can be used to create micro-CT datasets for 3D images, as well as quantitative measurements of individual vessels or of the entire 3D bed. Micro-CT has a lower image resolution, which may limit examination of the microcirculation. Thus SEM, with its higher resolution, is an important complementary method to employ.