Bioanalytical Techniques in Advanced Microscopy - Research Paper Example

The Accuracy of Scanning Electron Microscopy (SEM), Confocal Microscopy, and Atomic Force Microscope (AFM) in the Reconstruction of 3-D Models Name of student University / lab address Abstract Objectives The purpose of this review is to analyze the accuracy of scanning electron microscopy (SEM), confocal microscopy, and atomic force microscope (AFM) in the reconstruction of 3-D models. Key findings The AFM method offers a number of benefits for studying nanostructures in comparison to SEM tomography. AFM technique has the capability of scanning serial sections of optional thickness without a need for the beam of electrons. The confocal microscope has the capability of creating sharp optical sections, making it possible to construct 3-D renditions of the sample. Summary SEM, AFM, and confocal microscopes are specialized devices that allow scientists to view microscopic subjects. AFM involves imaging a specimen by the use of a cantilever tip or probe, having a radius of 20nm. The SEM is a device that creates a hugely magnified image by making use of electrons rather than light to build an image. In a confocal microscope, a single point of excitation light is scanned over the sample. AFM technology is not popular for imaging nanostructures and reconstruction of their 3-D structures. However, the AFM method offers a number of benefits for studying nanostructures in comparison to SEM tomography. Confocal microscopy is normally employed where 3-D structure is crucial. Key words Scanning electron microscopy (SEM), confocal microscopy, atomic force microscope (AFM), and 3-D reconstruction The Accuracy of Scanning Electron Microscopy (SEM), Confocal Microscopy, and Atomic Force Microscope (AFM) in the Reconstruction of 3-D Models Introduction Microscopy can be described as a technical field that uses microscopes to view objects and samples that may not be viewed with naked eyes. It is not possible to see such objects because they are normally not in the resolution range of the normal eye. Microscopy has three widely recognized branches namely: scanning probe, electron, and optical microscopy[Kam05]. Electron and optical microscopy concern the refraction, reflection, or diffraction of electron beams or electromagnetic radiation beams interacting with the specimen/sample, and the sequent collection of another signal or the scattered radiation ready to create an image. This process can be performed through wide-field irradiation of the specimen (for instance transmission electron microscopy and standard light microscopy) or by scanning of a fine beam across the specimen (for instance scanning electron microscopy (SEM) and confocal laser scanning microscopy). Scanning probe microscopy concerns the interaction of a scanning probe with the surface of the sample one is interested with. Advancement in microscopy has revolutionized biology and continues to be a crucial technique in physical and life sciences[Hit13]. This paper will focus on three microscopy technologies namely scanning electron microscopy (SEM), confocal microscopy, and atomic force microscope (AFM) and their capabilities of generating 3-D models of nanostructures. This will be achieved by reviewing the available literature on comparison of microscopy techniques for the 3-D model generation of nanostructures. Each of the three microscopes has its own advantages and disadvantages, and each needs a varying technique for obtaining 3-D information from the scans carried out[Qia07]. This review is specifically focused on evaluating the effectiveness of three potential techniques of 3-D generation and inspection. The SEM, confocal microscopy, and AFM, all permit users to reconstruct 3-D information of MEMS (micro-electro-mechanical systems) devices. This review will analyze the accuracy of each microscope in the reconstruction of 3-D models. This will be achieved by comparing the three microscopes against each other, to determine their weaknesses and strengths. Reconstructing 3-D images at nanoscale needs considerations and specialized devices that are not necessary when generating 3-D data at large scales[Zol13]. SEM, AFM, and confocal microscopes are specialized devices that allow scientists to view microscopic subjects. Each of these devices has specialized physical properties that can impact the manner in which 3-D data is collected. In such small scales, an image from an AFM microscope will display artifacts if the structures or features being evaluated are lesser than the tip itself. SEM images are generated by employing a concentrated beam of electrons, and on some samples, charging effects produced by concentration of electrons may look like a white blooming regions. A confocal microscope uses lasers for imaging, and their wavelengths permit accurate measurements in the nanometers. By the use of a scanning point of light rather than a full sample illumination, confocal microscopes produce a little higher resolution, and substantial enhancement in optical sectioning. Therefore, confocal microscopy is normally employed where 3-D structure is crucial[Rez06]. Methods Atomic force microscopy is one of the most potent devices for finding out surface topography at subnanometer resolution[Che05]. It is one of the best devices for manipulating, measuring, and imaging matter at the nanoscale. It is a crucial device for studying biological specimens because of its capability of imaging surfaces under liquids. AFM involves imaging a specimen by the use of a cantilever tip or probe, having a radius of 20nm[Che05]. That is, t operates by physical interaction of the molecules on the cell surface with the cantilever tip. The cantilever tip is normally placed some nanometers above the surface employing a feedback mechanism that evaluates tip-surface interactions. Adhesion forces between the cell surface molecules and the cantilever tip are detected as cantilever deflections. Scanning of the tip is done repeatedly over the sample while variations in tip height are recorded to produce a topographic image of the surface. Precise scanning is enabled by piezoelectric elements that enable tiny but precise and accurate movements on electronic. Therefore, the cantilever tip may be used to examine living cells’ single molecular events in psychological conditions, and may be employed to image live cells with atomic resolution. The AFM can be operated in several modes, on basis of the application. Generally, probable imaging modes are split into static modes and several dynamic modes, where the vibration of the cantilever is done. Presently, this is the only microscopy method available that directly offers functional, mechanical, and structural information at high resolution[Hit13]. Confocal microscopy has a number of advantages as compared to conventional optical microscopy, including the capability of collecting serial optical regions from thick samples, eliminating the image degrading out-of-focus information, and having controllable depth of field[Sie04]. The fundamental principle in this microscopy technique is using spatial filtering to remove out-of-focus flare or light in samples that are not thin than the focal plane; thus, increasing optical contrast and resolution of a micrograph. This technique also allows the generation of 3-D structures from the obtained images[Rez06]. It was named confocal microscopy because the pinhole of the microscope is normally conjugate to the focal point of the lens, therefore it can be referred to as a confocal pinhole. It has gained popularity in industrial and scientific fields, and general applications are in material science, semiconductor inspection, and life sciences. In a confocal microscope, a single point of excitation light is scanned over the sample. This point is a diffraction limited spot on the sample and is created by either focusing a parallel laser beam or by imaging an illuminated aperture located in a conjugate plane to the sample. Having only one point illuminated, the illumination intensity quickly falls off below and above the focal plane as the beam diverges and converges, therefore minimizing excitation of fluorescence for intrusive objects located out of the plane of focus being evaluated. Fluorescent light passes through a dichroic reflector and then through a pinhole aperture located in a conjugate plane of focus to the sample. Whatsoever light emanating from sections away from the illuminated point is blocked by the aperture, therefore offering additional attenuation of out-of-focus interference[Sem05]. A photodetector detects the light passing through the image pinhole. Typically, a computer controls the sequential scanning of the specimen and also assembles the image for presentation onto a video monitor. The SEM microscopy uses electrons rather than light to create an image. Since SEM microscopes were developed, they have helped researchers to evaluate a big number of specimens. The SEM has a number of advantages as compared to conventional microscopes. It has a very high resolution; therefore specimens that are closely spaced can be magnified at very higher levels[See04]. It also has a large depth of field, allowing more of a sample to be in focus on one occasion. Since SEM employs electromagnets instead of lenses, one has a lot of control in the level of magnification[Kub07]. As a result of these benefits, as well as the clear images produced by SEM, make SEM microscope to be one of the most important devices in research nowadays[Zol13]. The SEM is a device that creates a hugely magnified image by making use of electrons rather than light to build an image. An electron gun is used to produce a beam of electrons at the top of the microscope. In the microscope, the beam of electrons travel through a vertical path, that is placed within a vacuum. The electron beam travels through lenses and electromagnetic fields, which focus the electron beam down toward the specimen. When the electron beam strikes or come into contact with the specimen, X-rays and electrons are ejected from the specimen. Then, detectors collect these secondary electrons, backscattered electrons, and X-rays and convert them into a signal that is sent to a video monitor. This creates the final image. Discussion A number of studies have already compared the results of different microscope systems including AFM, SEM, and confocal microscopy[Pud94]. However, few studies have been conducted on combination of different modalities into a single reconstruction[Kam05]. There are few studies on the AFM capabilities of reconstructing 3-D models of nanostructures[Kam05]. AFM technology is not popular for imaging nanostructures and reconstruction of their 3-D structures. However, a few studies have compared SEM and AFM capabilities in imaging nanostructures and reconstructing their 3-D structures. SEM technique visualizes features on the surfaces of samples and produces a very good 3-D view. It produces results similar to the ones produced by the stereo light microscope. In the year 2011, its best resolution was 0.4 nanometer[Luc121]. Past studies have indicated that SEM tomography can allow someone to visualize and process 3-D reconstruction of nanostructures[Den04]. Other studies have indicated that this can also be done using AFM technology. The most common method of 3-D reconstruction in SEM is depth from stereo. The AFM method offers a number of benefits for studying nanostructures in comparison to SEM tomography. These advantages include: a) AFM technique does not cause any damage resulting from electron beam, vacuum, low temperature, high temperature, among others; b) AFM technique can be applied to cells of numerous sizes, while SEM cryotomography is presently limited to small cells. AFM technique assists someone to view structures of several sizes; c) AFM technique can be used to visualize serial sections of optional thickness[Rit10]. Depending on the aim of the research, AFM technique has the capability of scanning serial sections of optional thickness without a need for the beam of electrons. On the contrary, SEM needs multiple scattering events based on electron that can severely degrade quality of the image[Rit10]. It is worth noting that AFM imaging of thinner sections is very possible when advancement in technology will allow such thin sections to be cut. AFM examine the surface of a specimen by a sharp tip, situated at the free end of a cantilever. The cantilever is forced to deflect or bend by atomic forces between the specimen surface and the tip. While the scanning of the tip across the sample takes place, a detector quantifies the cantilever deflections and creates a map of surface topography. Hence, thin sections thickness on a substrate in general does not influence the AFM resolution in any way. This specific feature of AFM technique may help AFM to readily reconstruct 3-D inner structures at a nanoscale than SEM[Sch081]. Although AFM seems to have more advantages over SEM, it still has some limitations in some areas. Unlike SEM, it is not possible to apply multiple tilted views in AFM. Therefore, someone requires using so many serial sections of certain thickness to reconstruct an entire cell with a given thickness. Additionally, it is hard to determine the various cross sections of the same cell hiding amongst many cells at varying slices[Kam05]. Moreover, it is even quite challenging find better resolution of serial-thin-section images employing AFM technique. At present, AFM does not afford better resolution of thin sections as compared to SEM; however, the AFM resolution of molecules has surpassed that of SEM. Although a number of factors may lead to the relatively low resolution of AFM for thin sections, the convolution effect of AFM tips is the chief reason. This effect usually makes it hard to find the exact measurements of imaged structures, and has been considered as a great problematic issue since AFM was invented[Kam05]. This problem has been partly resolved through sharpening of the AFM tips. The confocal microscope has the capability of creating sharp optical sections, making it possible to construct 3-D renditions of the sample. Data collected at optical sections imaged at regular and short intervals on the optical axis are applied to build the 3-D reconstruction. Computer programs can merge the 2-D image to build a 3-D rendition. This technique can render depth-resolved slices through a 3-D object by rejecting most of the out-of-focus light through a pinhole aperture. The image is normally reconstructed serially. Confocal microscopy is a crucial tool for imaging 3-D samples because of its non-invasive and non-destructive nature which maintain such spatial information. Nevertheless, imaging challenges are still encountered; attenuation of the signal by the sample under examination, convolution by unknown point spread functions, and low signal-to-noise ratio contributes to low quality images or blurred images[Den04]. Microscopy systems with the capability of achieving the lowest depth of focus can attain the best accurate 3-D reconstructions. Multifocal techniques extract depth by assuming multiple images at various planes of focus, with reconstruction performed by examining in which image an area of the scene seems to be in the highest degree of focus. Through understanding the heights at which the focus was set over an image series, it is possible to assign depth to pixels depending on the image they are sharpest in. Confocal microscopy is particularly important in extracting depth from focus. The microscope is designed in such a way that the focal plane through a sample is very narrow on the axial axis, and hence, depth information extracted is very accurate. Hence, confocal microscopy is normally employed where 3-D structure is crucial[Kam05]. There are a number of methods that are available for extracting the depth information and creating a total 3-D model from it. A study conducted by Pudney et al. (1994) evaluated techniques for the detection of surfaces in confocal microscopy[Pud94]. The capability of detecting feature types helps in the reconstruction of 3-D of the optical slices. In the method by Pudney et al., feature detectors were computing local energy by convolving images with oriented, quadrature pairs of 3-D filters. More accurate confocal images can be restored by using the fast Fourier transform to carry out convolution and iteratively works. There is also a method that uses the maximum likelihood technique to optically section images better. One advantage of using maximum likelihood techniques is that they optimize the images under the constraints of Poisson noise. A number of methods of de-convolution are utilized as a way of increasing the confocal axial resolution. These techniques enhance the accuracy by improving the information at lower light levels, and more precisely by finding out the parts of the image which actually represent the present plane of focus[Ras05]. Conclusion SEM, AFM, and confocal microscopes are specialized devices that allow scientists to view microscopic subjects. Each of these devices has specialized physical properties that can impact the manner in which 3-D data is collected. AFM involves imaging a specimen by the use of a cantilever tip or probe, having a radius of 20nm. The SEM is a device that creates a hugely magnified image by making use of electrons rather than light to build an image. In a confocal microscope, a single point of excitation light is scanned over the sample. This point is a diffraction limited spot on the sample and is created by either focusing a parallel laser beam or by imaging an illuminated aperture located in a conjugate plane to the sample. AFM technology is not popular for imaging nanostructures and reconstruction of their 3-D structures. However, the AFM method offers a number of benefits for studying nanostructures in comparison to SEM tomography such as not causing any damage resulting from electron beam, vacuum, low temperature, high temperature, among others; it can be applied to cells of numerous sizes, while SEM cryotomography is presently limited to small cells; and it can be used to visualize serial sections of optional thickness. The confocal microscope has the capability of creating sharp optical sections, making it possible to construct 3-D renditions of the sample. Data collected at optical sections imaged at regular and short intervals on the optical axis are applied to build the 3-D reconstruction. Hence, confocal microscopy is normally employed where 3-D structure is crucial. 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