rmmcdonald's blog

    The impact of photobleaching on a specimen can be detrimental and irreversible. Depending on the fluorescent stained utilized, certain fluorophores photobleach at a more rapid rate than others requiring more care. The time lapse images collected from part I reveal the different properties of each fluorophore. Admittedly, the photobleaching rate constants extracted from the trend lines of Figure 4, Figure 5, and Figure 6 suggested unexpected values. Since each of these rate constants were so similar, I felt that no definitive conclusions may be drawn about comparing each of the fluorophores with this data. In future experiments, I will try to increase the number of significant digits in the data collected and increase the overall quantity of numerical data. Therefore, I utilized the mostly qualitative data to reach the following conclusions.

Visually, DAPI appears in Figure 1 to decrease less in intensity than the fluorophores seen in Figure 2 or Figure 3. Furthermore, the overall trend line of the DAPI stained nuclei in Figure 4 exhibits the smallest rate of photobleaching. In comparison, Figure 6 and Figure 7 suggest that the rhodamine stained F-actin and FITC stained tubulin have the same rapid rate of photobleaching. If more trials were conducted then we could have conclusively stated which fluorophore photobleached the fastest. However from these varying trend lines and visual differences, the data would suggest that different fluorophores bleach at different rates. Due to DAPI’s chemical structure it might be less likely to bind to neutral compounds like oxygen that inhibit photon emission. Conversely, Rhodamine and Fluorescein may act as unstable compounds that prefer to bind to other molecules than undergo periodic photon emission and absorption.

A Nikon Inverted Optical microscope at 40x magnification was utilized throughout all experiments. For studying the rate of photobleaching of DAPI stained nuclei, we set the exposure time 600 ms and a previously untouched area of cells was exposed to constant fluorescent light for 5 minutes. During those 5 minutes, 31 photos were automatically captured at 10 second intervals and the results are visualized by Figure 1. The brightness of each nuclei clearly decreased and the images blurred overtime as evident in Figure 1.

We repeated this process to capture the rate of photobleaching of fluorescein stained tubulin. Once we located an unbleached area of cells, we set the exposure to 2,000 ms and begun the time lapse video. Figure 2 represents the specimen over the course of 5 minutes of constant exposure. The decrease in intensity in Figure 2 seems more evident than in Figure 1, clearly exhibiting effects of photobleaching.

Finally, we captured evidence of photobleaching of rhodamine stained F-actin. We initially set the exposure time to 8,500 ms, but found that it was difficult to visualize those results. Therefore we completed the experiment again with a lower exposure time of 7,500 ms. Figure 3 visualizes the results from the second trial of photobleaching the sample. Figure 3 reveals a more rapid decrease in intensity as picture B and C look relatively similar, unlike those time points from Figure 1 and Figure 2. 

Photobleaching is an unavoidable phenomenon in fluorescent microscopy that occurs when a fluorophore in a fluorescently labeled specimen forms a covalent bond with another molecule. As a result the fluorophore can no longer decay to its original state and emit a photon. In this lab we tested for the rate of photobleaching under various conditions and with three types of fluorescent stains: DAPI, fluorescein, and rhodamine. The intensity of nuclei were calculated over varying exposure times and were analyzed. We concluded that the use of an automatic shutter and excitation filters greatly reduced the rate of photobleaching for all fluorescent stains. Our data also suggested that DAPI photobleached at the lowest rate compared to the other fluorophores. Overall, fluorescent microscopy is an efficient way to visualize specific targets given that proper precautions are taken to prevent photobleaching.

Bozzone, Donna M. “Feeding Behaviors in Cellular Slime Molds: A Microbial System to Study Competition.” The American Biology Teacher, vol. 59, no. 9, 1997, pp. 565–572. JSTOR, www.jstor.org/stable/4450384.

Coleman, Annette. The Quarterly Review of Biology, vol. 51, 1976, pp. 362–363. JSTOR, www.jstor.org/stable/2823102.

DNA replication is an important biological process that acts as a basis for cell growth and division. When somatic cell divides, the DNA must also replicate itself so that each daughter cell will contain identical DNA transcripts. This process begins at sections of the DNA called the origin of replication. These origins of replication tend to contain AT repeats due to the fact that Adenine and Thymine have a weaker bond making it easier for those two nucleotide to seperate. Helicase will separate the base pairs and establish the replication fork. Initiator proteins will bind to the origin of replication and recruit essential proteins for replication. DNA polymerase will be recruited by the initiator proteins like primase and begin elongation in the 3' direction. Following initiation, the DNA will elongate in a 5' to 3' direction due to the free hydroxyl group. The opposite strand, or lagging strand, must be replicated in fragments because DNA must be replicated 5' to 3'. These fragments are called  Okazaki fragments. Since DNA replication is initiated at many parts in the genome, it will be terminated a various points as well. Termination will be a result of a blocked replication fork.

I thought it was interesting how Roman culture and art parallels so directly to leadership and military expansion at that time. During the Age of Plunder, the amount of new greek art greatly influenced sculptors at that time, bringing about a Hellenistic era of culture. Now, with the rise of Augustus the chaos of previous years of civil war have died down and the people of Rome can again switch their focus back to culture. Again Greek influence rises up, with Ovid's utilization of a specific form of Greek poetry. Although this style of Greek poetry had been in Rome for a while, Ovid repopularized it. I also find it ironic how during the time Greek culture was introduced that some Roman politicians were concerned about a moral decline. Ovid, who writes his work with Greek influence, often expresses scandalous ideas in his poetry, somewhat justifying politicians fears from a long time ago. 

DNA replication is an important biological process that acts as a basis for cell growth and division. When a cell must divide, the DNA must also replicate itself so that each daughter cell may contain identical DNA transcripts. This process begins by sections of DNA called origin of replication being targeted by intiator proteins. These origins of replication tend to contain AT repeats due to the fact that Adenine and Thymine have a weaker bond making it easier for those two nucleotide to seperate. Helicase will seperate the base pairs and establish the replication fork. Following intiation, the DNA will elongate in a 5' to 3' direction due to the free hydroxyl group. DNA polymerase will be recuited by the initiator proteins like primase and begin elongation in the 3' direction. The opposite strand, or lagging strand, must be replicated in fragments because DNA must be replicated 5' to 3'. These fragments are called  Okazaki fragment. Since DNA replication is intiated at many parts in the genome, it will be terminate a various points as well. Termination will be a result of a blocked replication fork. 

An economic analysis might seem like a rather uninteresting perspective to the antebellum era. However, the economic pressures that divided, yet united Northern and Southern America can not be overlooked. Early 1800s both North and South relied on farming. In the north, close family units worked together on a small subsistent farm with only a local trading economy available. In the South the farms were larger due to the climate and crops grown. Around 1810 the capitalist economy in the North started to take hold and break down the small family units. Woman began to move to the cities to work in factories and send back pay checks to the family. There was a sudden emergence of an unskilled labor, working class in the north. With the introduction of the cotton gin, the south underwent a smiliar explosive economic expansion. The number of cotton that could of been produced exponentially increased by 1840 and therefore the number of slaves required to operate such large operations increased as well. Even though the international slave trade was disbained in 1808, the upper south started to sell slaves to the lower south due to over crowding and industrailization in the upper south. Both the North and the South looked West as a means of economic expansion. The North needed the West to be strictly a free labor system so as to not compete with slavery. However the South wanted to move West with slaves in order to keep expanding their current reality. This results in the basis for initail conflict between the north and the south. 

Overall, each of the figures followed the same formatting style, with one large photo on the left and two smaller photos on top of each other to the right. However besides that similarity, most of the other characteristics of the complete figures are contrasting. The overall resolution and lighting of each of the photos were significantly different. The resolution of Figure 1 seemed blurrier and the lighting appeared overexposed compared to the sharp resolution of Figure 2. Figure 1 contained even, white spacing between each of the panels while Figure 2 had a more significant gap between panel B and C. This links to the sizing of B and C, where the panels in Figure 1 were horizontal in contrast to Figure 2 where the panels were vertical. The letters that marked each panel in Figure 1 were black letters with no background. In comparison, the letters that marked each panel in Figure 2 were black letters with a white square background and a black border. The overall ordering of the panels were switched, therefore A from Figure 1 matches with B from Figure 2.

In Junior Year Writing Class at the University of Massachusetts, Amerhst, Professor Brewer assigned a project to capture and replicate signs of phytophagy on the UMass campus. This project allowed students to study what controllable factors and unavoidable discrepancies appear when replicating an individual’s methods. Furthermore, capturing evidence of phytophagy on the UMass campus offered a process to learn about how to create a perfect figure and follow others’ methods. The focus of my figure was a leaf found on a large bush in the rooftop garden of the John W. Olver Design Building. I chose this specimen due to its convenient location and obvious signs of phytophagy. In addition factors were more easily controlled since this is a privately maintained garden therefore the bush would not be destroyed. The time of day, zoom, perspective, and quality of the photos were also considered when this example of phytophagy was captured so that it can be replicated accurately.