Fluorescence Microscopy and Cultured Animal Cells - Essay Example

?Laboratory report on Practical Fluorescence Microscopy Practical a & b Use and imaging of cultured animal cells Submitted By Bearing student no Your student number On Date For the Course (CELB40100) Summary: A laboratory work was done on fluorescence microscopy with an aim to get practical knowledge on the use and imaging of cultured animal cells. For this study, pre-cultured HeLa cells were counted and then plated on coverslips placed in culture dishes. The cells, attached to the coverslips, were washed with phosphate buffered saline (PBS) and fixed using chilled methanol or paraformaldehyde (PFA), depending upon the protein to be stained. The PFA fixed cells were permeabilised using triton-x 100 (TX 100). Cells were then incubated with the primary antibody, against the target protein, and washed thoroughly to remove the unbound antibodies. Then the cells were incubated with secondary antibody targeted against the primary antibody, and again washed with PBS. Cell nuclei were counter stained with Hoechst33342. Coverslips with the cells were mounted with Mowiol on clean glass slide and observed under a fluorescence microscope after hardening of the Mowiol. Using proper filters and the objectives, the target proteins were viewed and images were taken and stored. Specific acquisition parameters of the imagery were noted for the record. Introduction: Fluorescence microscopy utilizes two facts: one that an antibody binds to an antigen and second, that fluorescent dyes or fluorochromes emit light of a longer wavelength when irradiated with short wavelength light (Herman, 1997). Only some proteins (Mocz, 1999) have this inherent property possessed by fluorochromes; others have to be rendered fluorescent by tagging them with fluorochromes. For this purpose the protein of interest is first bound by a primary antibody against that protein and then a secondary antibody tagged with a fluorochrome is bound to the primary antibody. This fluorochrome is excited in the microscope and can be seen by naked eyes. To visualize a protein of interest to as close to its natural state as possible, the cells are first fixed using a fixative. There are two types of fixatives: crosslinking and precipitating. The former is done by aldehydes and the latter by alcohols. Crosslinking fixatives act by creating covalent chemical bonds between proteins and anchors soluble proteins to the cytoskeleton (He, 2011). Precipitating fixatives act by reducing the solubility of protein molecules (Bacallao and Stelzer, 1989). The duration of fixation is very important; over fixation will increase the background noise and the full specimen will appear stained giving false positive results (Burns, Cuschieri, and Paul, 2006). Since the cells are fixed prior to staining, they may become impermeable to antibodies. In such situations, the specimen has to be made permeable to the antibodies using detergents like Triton X-100, and saponin (Koley and Bard, 2010). Also, not all target proteins are readily accessible to the antibodies; in such cases the specimen needs another treatment called antigen retrieval or antigen unmasking. This procedure involves boiling the sample in solutions like sodium citrate at certain pH (Ino, 2003) or incubating the specimen with enzymes like trypsin or pepsin (Shi, et al., 1993). Some specimen have inherent autofluorescence or fluorescence acquired by PFA; in such cases the autofluorescence needs to be quenched by a process called quenching. This is done by treating samples with glycine or sodium borohydride (Baschong, Suetterlin, and Laeng, 2001). Methods: A tissue section, cryosection or cells can be used to visualize the protein of interest. When cellular proteins are to be seen, the cells have to be grown on transparent surface like coverslips or dishes. For this particular exercise, HeLa cells were stained for ?-tubulin and nuclei were counterstained. A brief methodology is given below: Sub-culturing HeLa cells: A cell culture dish was provided with HeLa cells growing in it. Media was aspirated from the dish and the cells were rinsed with 2 ml trypsin-EDTA. The cells were then incubated with fresh 3 ml trypsin-EDTA for about 4 min in a humidified CO2 incubator to detach the cells from the dish surface. Constant monitoring of cells is important, as over exposure to trypsin is not favourable for the cells. When the cells detach from the culture dish, they start floating. As soon as all the cells started floating, 7 ml of growth media was added and the entire content of the dish was transferred to a 50 ml tube. 10 ul of the cells were aspirated from the tube and transferred to a Neubauer chamber and the cells were counted. The cells in the tube were then appropriately diluted with growth media to attain a dilution of about 60,000 cells/ml. 60,000 cells were then transferred to a culture dish containing a sterile coverslip on the bottom. Well spread cells were then incubated in the humidified CO2 incubator until the desired confluency was attained. Aseptic conditions were used while handling the cells. Methanol fixation and Staining of the cells: The coverslip with the cultured cells was washed with PBS and incubated in chilled methanol at -20 °C for 4 minutes. Cells were again thoroughly washed with PBS. About 75 ul of diluted mouse anti-tubulin antibody was placed on a piece of parafilm, and the coverslip inverted over it with cells facing downwards. This was incubated at 37 °C for 30 minutes. After incubation, the coverslip was thoroughly washed with PBS to remove the unbound antibody. Next, 75 ul of the anti-mouse antibody was spotted on the parafilm and the coverslip inverted over it. Again this was incubated for 30 minutes at 37 °C. Excess of secondary antibody was washed off using PBS. To counterstain the nuclei, 10 ul of Hoechst33342 was added and washed off after 5 minutes. To mount the coverslip, 7.5 ul of Mowiol was placed on a clean glass slide and the coverslip with cells facing downwards, was carefully put on it. All efforts were made to prevent air bubbles from trapping. The slide was stored in dark until Mowiol solidified and was then observed under a fluorescent microscope. The slides were first observed under 10X objective, acquired at 50 or 100 and photographs were taken. Same procedure was followed for 40X and 60X objectives. For students who performed PFA fixation, the coverslip was dipped in 3% PFA for 20 minutes, washed, and then quenched using 30 mM glycine, pH 7.4, for 5 minutes. After washing in PBS, the cells were permeabilised with 0.1% Triton-X 100 and stained as above. Results: The cell count vs dilution: the average cell count =31+38+25+18/4= 28 C1 = n x 10(4) = 28 x 10(4) = 280,000 cells/ml 60,000 x 13= 280,000 x v v1= 2.78 so, 13 - 2.8 = 10.2 (media was added for cell dilution) In general, the HeLa cells were adherent to the coverslip in monolayers because there was no overlapping of nuclei seen. They were present as different colonies (Figure 1). Several colonies were seen beside each other. The cells had typical elongated shape. The nuclei were irregular and oblong. The ?-tubulin appeared like “nests” around the nuclei (Figure 2). There was no regular alignment of ?-tubulin in the cells, but rather a dense and random network around the nucleus. However this randomness was lost in dividing cells as can be seen from figure 3. This is because ?-tubulin forms the mitotic spindles during cell division, which can also be seen in the figure 3. It can also be seen that dividing cells stain more intense for DAPI than non-dividing cells. Figure 1: DAPI and ?-tubulin staining of HeLa cells. One full colony and three flanking colonies can be seen. A dividing cell (blue arrows) can be seen at 7’O clock position of the centre colony. Figure 2: Nest of tubulin around the nucleus Figure 3: Magnified view of DAPI and ?-tubulin during cell division. Mitotic spindles (blue arrow) can be seen in the lower dividing cell, while the non-dividing cells (above two) have random network of ?-tubulin. It can also be seen that dividing cells stain more intense for DAPI than non-dividing cells. Conclusion: Fluorescence microscopy is a very good tool to visualize proteins in cells or tissue sections. This is because a coloured protein can be better visualized against a dark background. Here the specimen itself illuminates unlike other light microscopy where the specimen is illuminated by an external light source. This makes it very easy to locate the protein of interest. Also two different proteins can be co-localised using two different primary antibodies. Such staining is very important when the prevalence, location, and amount of one protein is studied with respect to another protein. The amount of fluorescence is directly proportional to the amount of protein; hence protein amount can also be calculated using this method (Rost, 1991). The wide-field fluorescence microscope image includes information above and below the focal plane; hence protein location can be seen in 3-dimentions. This allows spatial and temporal relations of one or more proteins which is not available with other types of microscopy. Since antigen-antibody reactions are involved in this method, the method has inherent specificity and sensitivity. Using GFP-tagged proteins, even live cell imaging can be done using fluorescence microscopy. However, given the cost of the instrument and the chemicals required, not everyone can afford to buy and use this technology. Since all proteins are different from each other in more than one ways, all steps in the protocol have to be standardized individually. Steps like permeabilisation, antigen retrieval, quenching of autofluorescence, and blocking has to be standardized on one on one basis. Over-fixation in PFA can give background noise and will give false positive results. This calls for an efficient quenching process. Improper blocking step can give nonspecific results too. Also, there are many auto-fluorescent endogenous molecules in the cells; they can also give false positive results. One of the disadvantages of fluorescence microscopy is its inability to delineate cellular structures other than those that are immunostained (Suzuki, 1997) Also the, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are some other disadvantages of fluorescence microscopy (Merdes, Stelzer and Mey, 1991). Still being a light microscope, it does not have resolution like that of an electron microscope. Use of GFP to visualize a live cell can also give wrong information, as presence of GFP in the cells itself is abnormal, it can change the micro environment of the cells. To conclude, wise selection of antibodies, standardization and validation of the protocol, and expertise in the field can prove this technology to be very rewarding and efficient for a scientist. References: Bacallao, R., Stelzer, E.H., 1989. Preservation of biological specimens for observation in a confocal fluorescence microscope and operational principles of confocal fluorescence microscopy. Methods Cell Biol, 31, pp. 437-52. Baschong, W., Suetterlin, R., and Laeng, R.H., 2001.Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). J Histochem Cytochem, 49(12), pp.1565-72. Burns, J.M., Cuschieri, A., and Paul, A., 2006. Campbell Optimisation of Fixation Period on Biological Cells via Time-Lapse Elasticity Mapping. Jpn. J. Appl. Phys. 45 pp. 2341. Available at: < http://jjap.jsap.jp/link?JJAP/45/2341/ > [Accessed 18 February 2012]. He, Z., Campolmi, N., Ha Thi, B.M., Dumollard, J.M., Peoc'h, M., Garraud, O., Piselli, S., Gain, P., and Thuret G., 2011. Optimization of immunolocalization of cell cycle proteins in human corneal endothelial cells. Mol Vis, 17, pp. 3494-511. Herman, B., 1997. Fluorescence Microscopy. 2nd ed. New York. Garland Science. Ino H., 2003. Antigen retrieval by heating en bloc for pre-fixed frozen material. J Histochem Cytochem, 51(8) pp. 995-1003. Koley, D. and Bard, A. J., 2010.Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proc Natl Acad Sci USA, 107(39), pp.16783-87. Merdes A., Stelzer E.H., De Mey J., 1991. The three-dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy. J Electron Microsc Tech, 18(1), pp. 61-73. Mocz, G., 1999. Intrinsic Fluorescence of Proteins and Peptides. [Online] Available at: < http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/fluor.htm> [Accessed 2 February 2012]. Rost, F.W.D., 1991.Quantitative fluorescence microscopy. Cambridge. Cambridge University Press. Shi, S.R., Chaiwun, B., Young, L., Cote R.J., and Taylor, C.R., 1993. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem, 41(11), pp. 1599-604. Takeshi, S.,1997. DNA Staining for Fluorescence and Laser Confocal Microscopy. J Histochem Cytochem, 45, pp. 49–53. Read More