Discuss about the Macromolecular and Cell Biology
Electron microscopy
Max Knoll and Ernst Ruska of Germany discovered electron microscope in 1931 (Pease, 2013). Starting with the theoretical resolution of 10 nm the electron microscopes built in 1944 reduced to 2nm. Between 1930-1960, several modifications were made to finally build the electron microscope with better lenses, resolution improving and brighter electron guns for producing higher energy and velocity electrons for sample probing. The period 1960-2000 marks the innovation of different types of electron microscope such as scanning and Transmission electron microscope (Humphreys, Beanland & Goodhew, 2014).
The fundamental limitations of all the microscopes lay in the difficulty to probe structural details of cells with a given type of radiation as they are smaller than own wavelength. Optical microscopy lacks high resolution. It limited its use in many areas of scientific research although it was the first microscope used for studying structures of mitochondria and bacteria, which was set at the wavelength of visible light. However, smaller details could not be visualized by light microscope due to affects of the wave nature of light. Electron microscope eliminates the limitations of the light microscope by providing high-resolution image of the sample. In light microscope, optical lenses cannot obtain high magnification. Therefore, it is useful for the studying the biological ultra structures and discern even macromolecules.. It would not have been possible for biologists to view the submicroscopic cells organelles like endoplasmic reticulum, ribosomes, and centrioles or the internal structure of the organelles like mitochondria. Electron microscopy also made it possible to study virus and viroids (Spence, 2013).
The general principle of the electron microscope and the light microscope is same. Electron microscope uses the electron beams, as the source of illumination. Instead of light, high velocity electrons are allowed to travel in a vacuum tube. Electrons revolve around atomic nucleus and fly off from atom when excited by the heat energy. High voltage current is used to heat tungsten to release stream of electrons like light beam. A series of electromagnetic lenses are used to focus the beam of electrons. It works in contrast to the eye and objective piece lenses and condenser in the light microscope. The magnified image obtained from the object positioned between objective and condenser is visible on photographic plate or on fluorescent screen. However, on light microscope it is observed through eyepiece. The image obtained by electron microscope has shades of black, grey or white as electrons are colorless. Fluorescent screen is used to observe the final image as electrons cannot be viewed by naked eye (De Boer et al., 2015).
The wavelengths of the electrons are 100,000 times shorter than the visible light. Therefore, it has high resolution to detect details of smaller objects, even with small numerical aperture. The resolving power increases with the decreasing wavelength of light. For instance the, wavelength of the green light is 0.55µ. In comparison to the electron beam, it is 10000 times longer. The resolution of the electron microscope is maximum 0.005 nm theoretically. It is much smaller than the diameter of the single atom. The resolution of the light microscope is two million folds that of the naked eye. In electron microscope with the help of the electronic fields, high magnification is obtained. Consequently, the resolution of the electron microscopy and magnification practically is 200 times more when compared to the light microscope. Therefore, electron microscope plays a vital role in diverse areas of scientific research (Humphreys, Beanland & Goodhew, 2014).
View of Golgi apparatus
The significant advantage of the electron microscopy is its powerful magnification, which was developed for its use in scientific research fascinated by complex three-dimensional morphology. One of the applications of the electron microscopy is the study of the Golgi apparatus by Han et al. (2013).
Visualizing the Golgi apparatus was based on the principle of direct visualization of the biological material that is frozen hydrated by the electron microscopy. This helped detect the various structures in the native environment. The preparation method for Golgi apparatus is delicate as it is prone to fixation artifacts. Cryo-fixation method was chosen to better understand the morphology of Golgi and its function. Using this method the Guo & Jiang (2014) describes the possibility of arresting all the cellular processes. The dynamic process inside small cells and tissues could be arrested with this microscope. Han et al. (2013) analyzed the Golgi apparatus by the enclosing in the amorphous ice which can be processed by the freeze substitution method. It can also be analyzed using directly in hydrated state. It can be investigated using the tomography and the cryo-elctron microscopy. If not exactly like the native structure, the resin embedded sample can better reveal the previously unknown structural details.
The technique starts with simple negative staining materials on the electron microscope grids followed by plunge freezing of sample for its stabilization in vitreous ice. Here massive temperature drops all the processes in the sample, which then looks like glass in high viscosity. Cooling rate is reduced by the high-pressure freezing or plunged directly into cryogen. Later in fully hydrate state the sample is examined in cryo-electron microscopic. The resolution of biological specimen is determined to near atomic level of 0.33–0.46 nm and was considered recent improvement. However it was limited to only examination of viral capsid proteins. Similarly, single-electron counting detectors were successful for ribosomes and proteomes. However, it was not completely successful for Golgi apparatus. Yet the single particle analysis facilitated the understanding of the architecture of the membrane bound vesicles containing coats on their interaction with electron beam (such as Clatharin) as well as cisternal buds. This technique helped to understand the Sec13/31 COPII at (3 nm resolution) coat architecture which contains cytoplasmic coat proteins. This mediates the intracellular transportation of cargo and lipids. Further, the flexibility during formation of coat and its assembly was better determined by combining mass spectroscopy and cryo-electron microscopy (at 1.2 nm resolution). In short the cryo-fixation technique including the high-pressure freeze fixation combined with the freeze substitution allowed observation of the constricted tubules by COPII like beads on string (Han et al., 2013). Overall, it was possible by the electron microscopy to detect Golgi saccules after inducing the secretion of procollagen in vitreous ice-embedded samples
There are limitations to the cryo-fixation technique as the process of vitrification is not completely clear although it overcomes the limitations of previously used resin embedded technique. Vitrification of samples is however beam sensitive creating low signal-to-noise ratio. The limitation is high in case of mammalian cell culture with low cell number, where block surface for cutting may not have single cell (Bouchet?Marquis et al., 2008). Moreover, cutting artifacts make it difficult to analyze certain Golgi areas. Further, constantly marinating the sample at −140 °C will prevent flow of sample at timescale of a realistic experiment. Further limitation is the probability of the formation of ice crystal growth in case of lager tissues or cells (Dobro et al., 2010). Beam sensitivity of the vitrified sample is further limitation that requires good image processing software. The extent of the structural diversification in electron-microscopy due to minimal composition differences’ is not yet determined. Infact further modifications are needed to fixation and labeling technique to view the molecular interactions in Golgi despite so much advancement in electron microscopy, for better describing the secretory pathway in its native state (Li et al., 2013, Han et al., 2013). In conclusion, the decipher of ultrastructure of a functioning Golgi may be way to go as it may require good combination of high resolution light microscope with electron and cryo-electron microscopy.
References
BOUCHET?MARQUIS, C., Starkuviene, V., & Grabenbauer, M. (2008). Golgi apparatus studied in vitreous sections. Journal of microscopy, 230(2), 308-316.
De Boer, P., Hoogenboom, J. P., & Giepmans, B. N. (2015). Correlated light and electron microscopy: ultrastructure lights up!. Nature methods, 12(6), 503.
Dobro, M. J., Melanson, L. A., Jensen, G. J., & McDowall, A. W. (2010). Plunge freezing for electron cryomicroscopy. In Methods in enzymology (Vol. 481, pp. 63-82). Academic Press.
Guo, F., & Jiang, W. (2014). Single particle cryo-electron microscopy and 3-D reconstruction of viruses. In Electron Microscopy (pp. 401-443). Humana Press, Totowa, NJ.
Han, H. M., Bouchet-Marquis, C., Huebinger, J., & Grabenbauer, M. (2013). Golgi apparatus analyzed by cryo-electron microscopy. Histochemistry and cell biology, 140(4), 369-381.
Humphreys, J., Beanland, R., & Goodhew, P. J. (2014). Electron microscopy and analysis. Third Edition. London: CRC Press. ISBN: 9781482289343
Li, X., Mooney, P., Zheng, S., Booth, C. R., Braunfeld, M. B., Gubbens, S., … & Cheng, Y. (2013). Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature methods, 10(6), 584.
Pease, D. C. (2013). Histological techniques for electron microscopy. Elsevier. Retrieved from: https://books.google.co.in/books?hl=en&lr=&id=LCzLBAAAQBAJ&oi=fnd&pg=PP1&dq=Histological+techniques+for+electron+microscopy&ots=xM_9vDuPaF&sig=boRiSpzr0U_6SP0h-68uvf31GhY&redir_esc=y#v=onepage&q=Histological%20techniques%20for%20electron%20microscopy&f=false
Spence, J. C. (2013). High-resolution electron microscopy. OUP Oxford. Retrieved from: https://books.google.co.in/books?hl=en&lr=&id=PitoAgAAQBAJ&oi=fnd&pg=PP1&dq=electron+microscopy&ots=ecgzcU-1U8&sig=iBTIxl92p9_zky3cDg22jWLSQ10&redir_esc=y#v=onepage&q=electron%20microscopy&f=false
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