Fluorescent stained phalloidin was used to directly label the actin cytoskeleton of both fibroblasts (NIH 3T3) and LLC-Pk1 cells. Images of each cell type were taken at 10X and 100X magnification under a fluorescent filter, and in phase contrast. In visually examining the images, it is clear there is a stark difference in actin cytoskeleton composition for each cell type. Figures 3B and 4B demonstrate the actin cytoskeleton of LLC-Pk1 cells as a localized group of filaments around the nucleus and more compact. Figures 3D and 4D depict the cytoskeleton of NIH 3T3 cells under fluorescence, which appear to extend away from the nucleus and are less compact.
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Images of pig kidney epithelial (LLC-Pk1) cells that were treated with both primary (anti-tubulin) and secondary (goat anti-rat FITC) antibodies for indirect immunofluorescence were taken at 10X magnification (Figure 1B & 1D) and 100X magnification (Figure 2B & 2D) under a fluorescent filter. Additional images were taken in phase contrast at 10X and 100X magnification (Figure 1A & 1C, Figure 2A & 2C, respectively). Additional LLC-Pk1 cells were treated in the absence of a primary antibody, (with buffer solution and secondary antibody) to create a control group for indirect immunofluorescence. These cells were imaged at 10X magnification (Figure 1C & 1D) and 100X magnification (Figure 2C & 2D) under a fluorescent filter. It can be visually discerned that there is a large difference in localization of fluorescent dye and ability to visualize tubulin structures between the two conditions. Figure 1B demonstrates a clear localization of fluorophores to the cells, specifically of tubulin structures around the nucleus. Figure 1D shows contrasting visual data, where fluorophores are seen indiscriminately binding to cellular structures as well as resting on parts of the slide that contain no cells. These differences are again highlighted, showing the direct localization of dye to microtubules, and random dying of miscellaneous cellular structures at a higher magnification in Figures 2B and 2D, respectively. Additionally, quantification of fluorescence was measured for both control and indirect immunofluorescence groups for all magnifications. It is apparent that the control group possesses a lower fluorescence intensity in localized tubulin areas than the group using the primary antibody for indirect immunofluorescence at 10X magnification (22.123, 46.028, respectively) and 100X magnification (31.249, 63.962, respectively) (Table 1).
Indirect immunofluorescence staining and the direct labeling of components within the cell are two ways in which one can visualize parts of the cell that are otherwise too microscopic or transparent to identify. In this lab, we explore how primary antibodies impact efficacy of targeted cellular structure staining with respect to indirect immunofluorescence. Additionally, phalloidin coupled with fluorescent dye is used to directly label the actin cytoskeleton of two different cell types in order to visualize and investigate the organizational differences between the two cell types’ actin cytoskeleton structure.
A possible explanation for the discrepancies between figures 1A and 2A could be that the replicated photo was taken of a different bloom on the tree. This could have happened because there was a lack of explicit detail explaining exactly which bloom was photographed in the original figure. Additionally, the angle was not specified in the methods whether is was to the right or left side of the ceiling of the conservatory, which could have helped in taking a more exact photo of the bloom. Figures 1B and 2B do not appear to be much different from one another. There are slight discrepancies between the amount of leaves captured at the top of the tree, but many of the factors that could have resulted in major differences in the figure were controlled by the limited angle options due to the corner placement of the tree. Differences between figures 1C and 2C can be explained by the insufficient explanation of how to create state borders on the figure map website. The default world map setting does not include state borders, so a box must be checked in order for the borders to be seen. With this box checked, each individual state can be highlighted and Florida can be colored in.These directions were not explicit in the methods section, and this step was missed in the execution of the map making for the replicate figure. Additionally, the type of font was not detailed in the methods, resulting in a different font used to label the figures on the replicate.
Part A both figures holds the widest discrepancy between the two figures. Figure 1A shows a flower with a bloom pointed at a 45° angle with respect to the ceiling of the conservatory. Figure 2A depicts a bloom at a 90° angle with respect to the ceiling of the conservatory. Part B of both figures displays a little discrepancy between both figures. Part C both figures varies slightly with respect to the outlining of the states within the United States, and the highlighted portion of florida. Figure 1C displays a map in which the states of the United States are outlined with borderlines, and the state of Florida is highlighted in red. Figure 2C does not contain state borders within the United States, and the state of Florida is colored grey along with the rest of the United States. Additionally, differences in font are noted for the figure labels in each figure.
The goal of the Methods Project is to explore the importance of explicit, detailed and concise writing when composing a scientific paper. Scientific writing must reflect these qualities in order for replication of experiments and analyses to take place by other interested scientists. The methods project demonstrates how the smallest omission of important information can lead to large differences in results of an experiment. The figure created for the methods project details Calliandra haematocephala, the Powder Puff Tree, and its indigenous locations throughout the world. The exhibit of the Powder Puff Tree at the University of Massachusetts is very easy to spot, and contains only one tree with a very small number of blooms. For these reasons, this tree was believed to be a one of the best options on campus to complete this project with accurate replication. The limited number of blooms offered little room to choose an incorrect flower, and the corner placement of the tree in the conservatory made it very simple to photograph the tree from the same angle. Additionally, the limited amount of countries the tree is found to be indigenous in limits the amount of error in missing a country on the map portion of the figure. Controlling for these factors allows for a more accurate and analogous replication for the figure of Calliandra haematocephala.
The original and replicate figures created of Calliandra haematocephala possess qualitative differences despite having the same methods followed in order to create them. Figure 1A and 2A are photos of different blooms on the tree, resulting in a large visual difference in the figure. Figures 1B and 2B are of the trunk and branches of the tree, and appear to be the most alike between all three components of the figure with only very slight differences in branches captured. Figures 1C and 2C also contain distinct differences. The borderlines for the states within the United States of America are outlined, and Florida is filled in red in figure 1C. Figure 2C does not possess borderlines for the states of the United States, and the state of Florida and the United States is a gray color. Additionally, different fonts were used to label each figure.
Overall, this laboratory exercise demonstrated the major elements that effect imaging in fluorescence microscopy. The net local concentration and degree of overlap of fluorophores and amount of the fluorescent dying in certain areas greatly effects the ability to achieve a bright and clear fluorescent microscopic image. Additionally, important microscope parameters such as the neutral density filters, and shutter/exposure time of the sample can greatly affect the brightness of an image and the rate at which fluorescent light decays over time, which is important to control in order to uphold the integrity of a sample.
The rhodamine labeled f-actin did not appear in the composite image of the three different fluorophores due to the intensity of the fluorescence being too low to register. This could be due to photobleaching that had previously occurred in this specific area of the sample, or if the sample was not labeled adequately with enough fluorescent dye. Time-lapse data for all three fluorophores under the same condition revealed discrepancies in rate of decay and initial intensity for each fluorophore. A relative high initial intensity for DAPI labeled dsDNA can be explained by the relative high net local concentration of bright fluorophores. Each nucleus contains a high concentration of dsDNA, which when stained with DAPI, creates a large solid fluorescent region with overlapping fluorophores. This differs from both tubulin and f-actin, which are of tube like nature, and appear as porous regions of interest where background light can seep through and be analyzed, making the initial brightness in the region of interest inherently darker. This difference is also reflected in the rates of decay, where in samples where there is less light to be diminished, the rate of decay appears much slower as it approaches the plateau of photobleached darkness. Time-lapse data for fluorescein under stained tubules under different conditions demonstrated discrepancies in rate of decay and initial intensity for all three conditions. The relative low rate of decay and low initial intensity for the images taken with a neutral density filters and auto shutter can be explained by the effect of the neutral density filter. The collective effect of the neutral density filters decreases the intensity of the epi-illumination light path by a factor of 32. This in turn lowers the ability for the fluorophores to be excited, causing a low initial intensity and low excitation, which leads to a lower rate of decay. The auto shutter condition displays a lower rate of decay compared to the open shutter condition due to the lower exposure to light over time. When the shutter is left on automatic, the sample is only exposed to light during the time an image is snapped as opposed to the entire duration of the time lapse. This lessens the time window that fluorophores can covalently bond to oxygen and other elements, and less brightness is lost over time. The open shutter condition leaves the sample exposed to the epi-illumination light path during the entirety of the time-lapse, allowing more time for covalent bonds to form with fluorophores and thus lose more brightness intensity over time. This explains the high relative rate of decay.
Fluorescein labeled tubulin was then time-lapse imaged under several conditions: auto shutter, open shutter, and open shutter with neutral density filters. Data was analyzed graphically for all three conditions (Figure 3). Open shutter data was shown to have the highest initial intensity value and rate of decay (Table 2). Auto shutter data contained midrange values for both initial intensity and rate of decay (Table 2). Time lapse data with neutral density filters and an open shutter demonstrated the lowest values for initial intensity and rate of decay (Table 2). Selected time lapse images visually demonstrate the difference in excitation intensity over time during the photobleaching process under all three conditions (Figure 4).