Techniques and Results Achieved from Scanning Electronic Microscopy and Transmission Electronic Microscopy - Coursework Example

Techniques And Results Achieved SEM and TEM Name Institution Course Instructor Date Techniques And Results Achieved From Scanning Electronic Microscopy And Transmission Electronic Microscopy Introduction Electronic microscopes are extremely prevailing tools for the visualization of biological samples. They facilitate the viewing of cells, tissues, and organisms in extremely inordinate details. Nevertheless, electronic microscopes are not used to view live biological samples. The biological samples ought to go through multifaceted preparation procedure to assist the samples endure the atmosphere inside the microscope. The process used for preparation denatures the tissues and may interfere with the sample’s appearance (Krivanek et al, 2010, p. 572). It poses a problem to researchers who wish to visualize biological samples as to how the samples may be preserved so as to retain its original appearance while still with the capability to endure the environment inside the electronic microscope. There are two major reasons as to why live samples cannot endure the environment inside an electronic microscope which include: the strength of the electronic beam that is focused at the sample and the vacuum in the microscope environment. The electronic beam in the transmission electronic microscope (TEM) results to challenges for the live samples because of the high energy. The beam ought to have sufficient power to penetrate the sample and out in the opposite direction. The temperature may reach 1500c where the beam focuses the sample. This temperature is extreme for live samples to withstand. The scanning electronic microscope (SEM) utilizes low power electronic beams, but it is still extreme for live samples (De Abajo, 2010, p. 209). The vacuum in the electronic microscope is vital for proper functioning. With the exception of a vacuum, the electrons focused on the samples would be bounced when they hit air elements. Liquid water is a common component in biological samples and it evaporates instantly in a vacuum; hence, biological samples may vaporize. For effective visualization, biological samples ought to be fixed to prevent destruction by electronic beams and dried to prevent destruction by the vacuum. This paper seeks to analyze the different images observed and techniques during visualization of human red blood cells by use of TEM and SEM. Scanning Electronic Microscopy All the samples ought to be of the required size to suit the specimen chamber and are typically attached on a specimen frame referred to as stub. Biological samples ought to be entirely dry since the specimen compartment is at an elevated vacuum. The samples require chemical fixation to reserve and safeguard their structure. Fixation is often carried out by incubating the specimen in a solution of a buffer which is a chemical fixative. The most common buffer used includes glutaraldehyde which is at times mixed with formaldehyde and other chemical fixatives. Additionally, it may be used as a post fixative with osmium tetroxide. The fixed specimen then undergoes dehydration (Krivanek et al, 2010, p. 577). Air drying mainly results to the collapsing and shrinking; hence, drying is mainly achieved through replacing the water cells with organic solvents such as ethanol or acetone. The organic solvents are consequently replaced with transitional fluids such as liquid carbon dioxide. Alternatively, it may be achieved by the utilization of critical point drying. The liquid carbon dioxide is lastly eliminated while still in the supercritical form to prevent the formation of a gas-liquid border within the sample as it dries. The dry specimen is eventually loaded on the specimen chamber by use of an adhesive which include either epoxy resin or an electrical conductive double-sided cement tape in the sample as it dries (Bogner, Jouneau, Thollet, Basset and Gauthier, 2007, p. 390). The specimen is then sputter-coated with gold prior to its examination using the electronic microscope. Some SEMs have cold surfaces for cryo microscopy; cryo-fixation is utilized in low-temperature scanning electronic microscopy carried out on the cryogenically fixed specimens. Cryo-fixed specimens may undergo cryo-fracturing inn unique devices to expose the interior structures, gold coated, and relocated to the SEM cryo-compartment while in frozen state. The samples are not cut into thin cross-sections because visualization by use of SEM utilizes the three-dimensional structure (Vihinen, Belevich, and Jokitalo, 2013, p. 8). The gold coating results to a conducting sample which draws way electrons that bombard the sample; hence, preventing the ‘charging artefacts’ effect which give a false appearance of the sample. Typically, an electronic beam is thermionically produced by an electron gun which has tungsten wire cathode. Tungsten is mainly utilized in thermionic electron guns because its melting point is high and it has the lowermost vapor pressure amongst of all metals; hence, it allows heating for electron transmission. Additionally, it is cost-effective. The electronic beam’s power ranges from 0.2 keV to 40 keV and is directed by condensers to a region between 0.4nm to 5nm in diameter. The beam penetrates duo scanning coils in the electronic pillars generally in the last lens which bounces the beam in the opposite axis so as to scan in a raster manner over a quadrilateral region of the area under the sample. After interaction of the sample with the primary electron beam, the electrons lose power through randomized smattering and absorption. The magnitude of the interaction region is dependent on the electron’s landing power and the compactness of the specimen. The energy transmission from the electron beam to the sample reflects high-energy electrons through elastic and inelastic scattering and electromagnetic radiations which are detectable by use of specialized detectors. The beam current engrossed by the specimen is detectable and used in the creation of images of the distribution of specimen current. The signals are amplified by use of electronic amplifiers which are presented as deviations in illumination on a computer monitor (Lombardi et al, 2009, 585). The resultant image is from the dissemination map of the strength of the signal which is produced from the scanned region of the specimen. SEM magnification is regulated ranging from about 10 to 500,000 times. As it is the case in transmission electronic microscopy, magnification of the image in the SEM does not result from objective lens power. SEMs may be inclusive of condensers and objective lenses; however, their role is the focusing of the beam to the required region. SEMs may function without either the condenser or objective lens as long as the electron gun can produce beams with adequately small thickness. The magnification is highly dependent on the current focused on the opposite scanning coils which is the ration between raster on the specimen and the rater on the display apparatus. Majority of SEMS produce single value in each pixel which leads to the production of black and white images. Nevertheless, the images are colorized by the utilization of either true or false colors. The colorization is achieved by use of either color look up tables or feature-detection software. Transmission Electronic Microscopy Transmission electron microscopy refers to a microscopy technique where a beam of electrons is passed through an ultra-thin specimen. The beam of electrons interrelates with the specimen as it penetrates. There is formation of an image from the interrelation of electrons that penetrate the specimen. Eventually, the image undergoes magnification and it if fixated onto an imaging machine or a photographic film. The image be distinguished by use of a CCD camera. TEMs form contrast images at smaller magnification which results from the absorption of electrons by the specimen at high magnification, multifaceted wave connections regulate the image intensity which requires proficiency for examination of visualized images. TEMs imaging device is inclusive of a phosphor screen which is made from small Zinc sulphide particles. Additionally, an imaging recording device may be utilized which include YAG display coupled CCDs (Vihinen, Belevich, and Jokitalo, 2013, p. 10). TEM composes of various apparatuses which involve a vacuum system which acts as a travel medium for electrons. Also, it composes of electromagnetic lenses, and electrostatic surfaces which regulate the beam as required. The most utilized mode during visualization by TEM is the bright field imaging mode. This mode imagery contrast is achieved by occlusion and raptness of electrons in the sample. Thicker parts of the sample tend to appear dark whereas parts with no sample tend to look bright; henceforth the name ‘bright field’. The sample is prepared by use of a multifaceted procedure. The specimens are required to be of extremely small thickness. The electron beam undergoes ready interaction with the sample. The samples have to undergo the fixing process so as to withstand the vacuum environment of the microscope. Fixation of biological samples makes us of Uranyl acetate and plastic embedding (de Jonge. and Ross, 2011, p. 659). The thickness of the sample ought to be thin enough for easy penetration of the electron beam. The required thin sections of the specimen may be achieved by passing the specimen over an diamond edge to obtain minute thin sections. This process does not result to extreme deformation of the sample during sectioning; the use of other inorganic substances results to sample deformation. The sample is covered with a good conductor such as carbon so as to prevent the charge formation on the surface of the specimen. The coating thickness is very minute and ought to be in nanometer which is achieved by the utilization of sputter coating system. The stain used in the sample either engrosses electrons or diffracts sections of the electron beam which is else projected on the imaging device Rothen-(Rutishauser, Schürch, Haenni, Kapp, and Gehr, 2006, p. 4353). Observing the human red blood cells by use of SEM and TEM Whole blood consist various cell types found in the circulatory system. Red blood cells are the most plentiful cells in the whole of the blood system and they compose of about 50% of the volume in the blood. The proportion of the blood volume taken up by the red blood cells is referred to as hematocrit (de Jonge. and Ross, 2011, p. 700). The major role of red blood cells is the transportation of oxygen to all parts of the body and the transportation of carbon dioxide to the lungs for exhalation. Red blood cells are enormously elastic and distortion of their cell wall results to the spring of their inherent shape within a few minutes. The red blood cells hold the cell membrane into a particular concave shape which results to increased surface area to volume ratio which allows the penetration of the cells through blood capillaries. The blood cells were imaged by SEM and TEM. Samples were collected from donors and fixed with 2% buffered gluteraldehyde solution (de Jonge. and Ross, 2011, p. 700). The Red blood cells were separated from the other blood components by use of a centrifuge. The red blood cells have a high density compared to the white blood cells; hence, the white blood cells floated on the top. Washing and chilled followed and the cells were ready for the drying process. Biological specimens present challenges for visualization by use of electron microscopes because they are poor conductors and they have cytoplasmic water. The samples ought to undergo drying and make them conductors to prevent the formation of artifacts. For SEM visualization the specimens were washed using ddH2O and dried on aluminum foils on SEM specimen stubs. The fixing procedure resulted to a very rigid cell wall; hence, striking drying techniques were not necessary (Hoehn and Nicpon, 2010, P. 49). For TEM visualization, the specimen was put in buffer solution and then stained with osmium for imaging facilitation. The specimen was added to epoxy and allowed to harden in an oven. For the preparation of cross-sections of the sample, ultra microtome was utilized to slice 100 nm slices from the specimen implanted in the epoxy. The thin slices were picked by use of copper TEM grid. Samples ought to made conductors for imaging in the electronic microscopes. Thin layer of gold, not more than 6 nm was used to spatter the samples for SEM imaging (Hoehn and Nicpon, 2010, P. 49). For SEM imaging, the process utilized the secondary electron detection. A small hastening voltage of 3-5 kV was utilized due to the thin layer of gold used. High energy electrons would lead to low resolution images due to large interaction volume with the sample. The SEM imagery showed the three-dimensional structure of the red blood cell. SEM 3- Dimensional Structure of Red Blood Cells (Hoehn and Nicpon, 2010, P. 50). For the TEM, high hastening voltage results to high spatial resolution and the small thickness of the sample limits the interaction volume. The samples did not undergo gold coating; nevertheless, they were stained so as to have regions of high electron density for imaging. The sample was stained by use of Osmium which provided even staining. TEM Image of Red Blood Cell (Hoehn and Nicpon, 2010, P. 50). Electron microscopes produce extremely powerful magnification. Electron microscopy has wide range applications in the fields of quality control, research for medical and forensic science, and nanotechnology. The high resolution enables the interior visualization of samples and magnifies images at a much greater degree. The major disadvantages of electron microscopy include cost, magnitude, and it requires trained expertise for use. There is formation of imagery artifacts which lead to distortion of the original imagery. The electron microscopes require keeping in spacious areas and protect the machine from unintentional effects of electrons (Park et al, 2008, p. 13730). The microscopes require upkeep so as to regulate the voltage supply. The microscopes also require constant flow of water so as to prevent damage of specimens by the heat produced during the energizing of electrons. Expertise is vital so as to minimize the formation of artifacts during the preparation of the sample for visualization. The specimens are visualized in a vacuum as the air elements would deflect the electrons. Hence, difficult and lengthy techniques are required for sample preparation. The most recent advances allow the viewing of hydrated samples by use of an environmental scanning microscope; however, its application is still partial. The SEM can only visualize the surfaces of the specimen unlike the TEM which reveals the internal structures (de Jonge. and Ross, 2011, p. 702). Conclusion This paper has describes the different techniques utilized in the SEM and TEM and the different images observed from visualization of human red blood cells. Without microscopy, cytology would not exist. Electron microscopes are vital tools for the study of cell tissues for both plants and animals and are extremely significant tools for the study of ultrastructural assessment of prokaryotic cells. Electron magnification magnifies images at a significantly high magnification compared to the traditional light microscopes. Both the SEM and TEM require the viewing of samples in a vacuum. Biological samples are not able to withstand the vacuum in the microscope environment; hence, the specimens require fixing before viewing. However, the process of sample preparation may result to formation of artifacts which interferes with the actual image of the sample. Both SEM and TEM utilize distinct techniques. For TEM visualization, samples are sliced into thin cross-sections which is not the case in SEM. Bibliography Bogner, A., Jouneau, P. H., Thollet, G., Basset, D. and Gauthier, C. 2007. A history of scanning electron microscopy developments: towards “wet-STEM” imaging. Micron, 38(4), pp. 390-401. De Abajo, F. G. 2010. Optical excitations in electron microscopy. Reviews of modern physics, de Jonge, N. and Ross, F. M. 2011. Electron microscopy of specimens in liquid. Nature nanotechnology, 6(11), pp. 695-704. Hoehn, K., and Nicpon, E. 2010. Human Anatomy and Physiology. San Francisco, Calif: Benjamin Cummings. Krivanek, O. L., Chisholm, M. F., Nicolosi, V., Pennycook, T. J., Corbin, G. J., Dellby, N. and Pennycook, S. J. 2010. Atom-by-atom structural and chemical analysis by annular dark-field electron microscopy. Nature, 464 (7288), pp. 571-574. Lombardi, V. C., Ruscetti, F. W., Gupta, J. D., Pfost, M. A., Hagen, K. S., Peterson, D. L. and Mikovits, J. A. 2009. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science, 326(5952), pp. 585-589. Park, Y., Diez-Silva, M., Popescu, G., Lykotrafitis, G., Choi, W., Feld, M. S. and Suresh, S. 2008. Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum. Proceedings of the National Academy of Sciences, 105(37), pp. 13730-13735. Rothen-Rutishauser, B. M., Schürch, S., Haenni, B., Kapp, N. and Gehr, P. 2006. Interaction of fine particles and nanoparticles with red blood cells visualized with advanced microscopic techniques. Environmental science & technology, 40(14), pp. 4353-4359. Vihinen, H., Belevich, I. and Jokitalo, E. 2013. Three dimensional electron microscopy of cellular organelles by serial block face SEM and ET. Microsc. Anal, 27, pp. 7-10. Read More

The organic solvents are consequently replaced with transitional fluids such as liquid carbon dioxide. Alternatively, it may be achieved by the utilization of critical point drying. The liquid carbon dioxide is lastly eliminated while still in the supercritical form to prevent the formation of a gas-liquid border within the sample as it dries. The dry specimen is eventually loaded on the specimen chamber by use of an adhesive which include either epoxy resin or an electrical conductive double-sided cement tape in the sample as it dries (Bogner, Jouneau, Thollet, Basset and Gauthier, 2007, p. 390). The specimen is then sputter-coated with gold prior to its examination using the electronic microscope.

Some SEMs have cold surfaces for cryo microscopy; cryo-fixation is utilized in low-temperature scanning electronic microscopy carried out on the cryogenically fixed specimens. Cryo-fixed specimens may undergo cryo-fracturing inn unique devices to expose the interior structures, gold coated, and relocated to the SEM cryo-compartment while in frozen state. The samples are not cut into thin cross-sections because visualization by use of SEM utilizes the three-dimensional structure (Vihinen, Belevich, and Jokitalo, 2013, p. 8). The gold coating results to a conducting sample which draws way electrons that bombard the sample; hence, preventing the ‘charging artefacts’ effect which give a false appearance of the sample.

Typically, an electronic beam is thermionically produced by an electron gun which has tungsten wire cathode. Tungsten is mainly utilized in thermionic electron guns because its melting point is high and it has the lowermost vapor pressure amongst of all metals; hence, it allows heating for electron transmission. Additionally, it is cost-effective. The electronic beam’s power ranges from 0.2 keV to 40 keV and is directed by condensers to a region between 0.4nm to 5nm in diameter. The beam penetrates duo scanning coils in the electronic pillars generally in the last lens which bounces the beam in the opposite axis so as to scan in a raster manner over a quadrilateral region of the area under the sample.

After interaction of the sample with the primary electron beam, the electrons lose power through randomized smattering and absorption. The magnitude of the interaction region is dependent on the electron’s landing power and the compactness of the specimen. The energy transmission from the electron beam to the sample reflects high-energy electrons through elastic and inelastic scattering and electromagnetic radiations which are detectable by use of specialized detectors. The beam current engrossed by the specimen is detectable and used in the creation of images of the distribution of specimen current.

The signals are amplified by use of electronic amplifiers which are presented as deviations in illumination on a computer monitor (Lombardi et al, 2009, 585). The resultant image is from the dissemination map of the strength of the signal which is produced from the scanned region of the specimen. SEM magnification is regulated ranging from about 10 to 500,000 times. As it is the case in transmission electronic microscopy, magnification of the image in the SEM does not result from objective lens power.

SEMs may be inclusive of condensers and objective lenses; however, their role is the focusing of the beam to the required region. SEMs may function without either the condenser or objective lens as long as the electron gun can produce beams with adequately small thickness. The magnification is highly dependent on the current focused on the opposite scanning coils which is the ration between raster on the specimen and the rater on the display apparatus. Majority of SEMS produce single value in each pixel which leads to the production of black and white images.

Nevertheless, the images are colorized by the utilization of either true or false colors. The colorization is achieved by use of either color look up tables or feature-detection software.

Read More