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Techniques used to observe and study structures in an animal cell.
1. In this essay, I will be explaining and describing the different techniques used to observe and study the structures in an animal cell. Through illustrating the similarities and differences between the compound light microscope and the transmission electron microscope and a description on how microscope slides are prepared. I will explain the stages of cell fractionation and the features some organelle possess which allow for them to be separated easily by this method.
2. To begin, I will be discussing the similarities and differences between transmission electron microscopes and compound light microscopes. Firstly, the transmission electron microscope uses a beam of electrons to produce an image of the specimen as opposed to visible light rays which is used by the compound light microscope. This means that the compound light microscope forms coloured images of the specimen while the transmission electron microscope doesn’t, this is due to the fact that electrons do not produce coloured micrographs which can be considered a disadvantage because it becomes harder to distinguish between the different organelles and see their true colour. This is why scientist often use computer-generated false-colour electron micrographs to help them visually, a means of distinguishing between the shades of grey shown in the original micrograph.
Another difference is that the specimen must be ‘dead’ when being viewed under the transmission electron microscope, this is because the specimen is placed in a vacuum, as opposed to the compound light microscope which can have living specimen. However, in most cases in order for the specimen to be seen by the compound light microscope they must be stained, I will go into further detail as to how this is done later on in this essay. The transmission electron microscope samples are prepared by a more complex procedure; as the samples have to be “extremely thin,” “to make these, the specimen is supported in a resin block to prevent it from collapsing during cutting, and is sliced with a diamond or glass knife. The section is then impregnated with a heavy-metal stain” (Kent, 2013).
In general, the transmission electron microscope has significantly higher magnification and resolution in comparison to the compound light microscope. “The magnification of an object is how many times bigger the image is when compared to the object,” (Toole and Toole, 2015) (a level) and the formula used to calculate this is “magnification=size of image/size of real object” (Toole and Toole, 2015) The transmission electron microscope can have a “magnification of up to 1,000,000x” (Theydiffer.com, 2018) as opposed to the light microscope which has “magnification of up to 1,500x” (Theydiffer.com, 2018)
Resolution is defined as “the minimum distance apart that two objects can be in order for them to appear as separate items.” (Toole and Toole, 2015) the transmission electron microscope has “High resolving power of up to 0.0001µm” (Theydiffer.com, 2018) versus the “low resolving power, usually below 0.30µm” (Theydiffer.com, 2018) of compound light microscope. This means that only the bigger organelle such as the nucleus, cytoplasm and cell membrane can be seen under the compound light microscope. While the transmission electron microscope can see the smaller organelle such as mitochondria, Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum, lysosomes and nucleolus.
fig.1 This is my illustration of the difference in image shown by the two microscope, highlighting what can and what cannot be seen by each microscope. My depiction of a typical electron micrograph of an animal cell was inspired by the one found on the website I used, slideplayer.com.
4. One of the techniques I have used in the laboratory to make structures more visible under the microscope is to focus the lens, this means the specimen can be seen much clearer at a higher resolution. Another technique I used is switching from lens to lens to find the most suitable magnification for a specific specimen. In addition, to make the specimen more visible I also used stains such as methylene blue so that I can see the organelles more clearly. A coverslip is mounted to ensure the specimen doesn’t move.
A more intricate method of scientists use is the fixation method “is to preserve cells and tissue components in a “life-like state” and to do this in such a way as to allow for the preparation of thin, stained sections” (Rolls, 2018) This is so the scientist can, in a sense, catch the given sample in action.
5. The process of cell fractionation is used by scientists to extract specific cell organelle for biochemical study. During cell fractionation, the cells are broken up into their separate organelles. The process goes as follows; a sample tissue is put in a cold, buffered isotonic solution. The sample is kept cold so that no enzymes can function and undergo reactions within the sample. To ensure the pH doesn’t fluctuate the solution is buffered. The solution is also kept under isotonic condition so that the water potential is the same inside and outside of the sample, this prevents the cells in the sample from bursting or shrinking due to differences in water potential.
Homogenisation is the step where the sample is put inside a homogeniser. A homogeniser, in Layman’s terms, is an elaborate blender used to break the sample in order to liberate the organelle. The organelles are now freely flowing in a solution known as the homogenate. The homogenate is “filtered to remove any complete cells and large pieces of debris.” (Toole and Toole, 2015) This is so the scientist can study the specific organelles they’re researching.
Ultracentrifugation happens after homogenisation. This is when the homogenate is spun at high speeds in a centrifuge, this is to separate the organelle according to their weight. The filtrate spun at slow speed, this is when the heaviest organelle are forced to the bottom which forms a pellet, for example, the nucleus. The supernatant is removed and transferred to another tube, leaving the pellet inside the tube. The supernatant is spun at a higher speed, again, the heavier organelle such as the mitochondria are forced to the bottom of the tube. This process is continued, the supernatant is removed again, however, at each time it is spun at a higher speed than the last.
The fact that each of the organelle have different weight makes them easier to separate by ultracentrifugation as each time the sample is spun, organelle can be separated and not mixed. Cell fractionation allows for scientist to look at each of the organelle in a cell separately and learn more about them.
6. To conclude this essay on the techniques used to observe and study structures in an animal cell, I have also briefly explained the methods I have previously used in a laboratory to produce better microscope images. I have explained the similarities and differences between compound light microscopes and transmission electron microscopes and highlighted the difference in the animal cell when viewed under both. I have described and explained the process of cell fractionation and the features of organelles that make them easier to separate using this method.
- Kent, M. (2013). Advanced biology. 2nd ed. Oxford: Oxford University Press.
- Theydiffer.com. (2018). Light Microscope vs Electron Microscope – Difference Between. [online] Available at: https://theydiffer.com/difference-between-a-light-microscope-and-an-electron-microscope/ [Accessed 17 Nov. 2018].
- Rolls, G. (2018). Process of Fixation and the Nature of Fixatives. [online] Leica Biosystems. Available at: https://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives/ [Accessed 17 Nov. 2018].
- Slideplayer.com. (2018). SlidePlayer – Upload and Share your PowerPoint presentations. [online] Available at: https://slideplayer.com/slide/5707909/ [Accessed 17 Nov. 2018].
- Toole, G. and Toole, S. (2015). AQA biology A level. 2nd ed. Oxford University Press.
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