The End of Summer

Last week, I finished my work at Princeton. Although it is not the ending of our project, the progress we made this summer is really important for the whole project.

The main part of our improvement includes two parts: thylakoid extraction and photosynthesis efficiency testing.

For the first part, the main procedures include: 1. using a lab blender to blend the mixture of alginate leaf pieces with grinding buffer; 2. putting the solution into the centrifuge for the pellet; 3. putting the pellet into the washing buffer and do the resuspension; 4. go through few more times of centrifuge to get the concentrated thylakoid.

For the testing, the main procedures include: 1. adding 0.10 mL of the suspension to 10 mL of 80% acetone in a test tube; 2. inverting the solution several times and then filtered through a Whatman filter paper into a large cuvette using a 50 mL glass funnel; 3. measuring the absorbance of the green solution using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. 

We tried multiple methods to see which works the best. There was failure sometimes, but we got through it and kept on moving forward. This project includes a lot of brainstorming and testing. Different from my last project, I have experienced more about the beginning part of a science project, including proposal writing, idea design and other preparation.

Since the project is still in progress, I will go back to Princeton to continue my lab experience during the school year. It is the consistency that makes a difference. 

Tissue Morphodynamics Lab: Weeks 9-10

Hello, this Danny from Dr. Nelson's Lab in Princeton, and its been quite a while since my last post. I been done for about a week now and I've just finished up organizing my data and images. The last two weeks at the lab were exciting and busy, as many of the graduate students and post-docs were preparing posters and presentations. Although Amira and Sriram seemed busy with experiments throughout the week, they still both made time to continue helping me with my research. 

During the ninth week, I was mainly focused on dissecting day 4, day 5, day 6, day 7 chicken lungs in order to create a morphology chart for my upcoming poster presentation. Ideally, I wanted to get the dissection done in the late morning, but if the microscopes were taken, I usually resorted to tracking cells on Imaris. On Imaris, I needed to track squares with higher densities and more cellular divisions per 12 hours, because Sriram and Dr. Gleghorn wanted to see if the cells illustrated similar rotational patterns to previous research done on rotational axes. There were on average 20 to 30 more divisions per data set in the higher density than the normal density data sets. After I finished cell tracking, the data was then analyzed through MatLab and saved. We also tracked cells in a worm structure, rather than in a square structure to see how different structural shape would affect the division of cells. The results were similar to the square, as the cells divided parallel to the edges of the structure.

The last week at the lab, there was a research symposium similar to the one EXP went in the Fall, that highlighted Physics in Living Systems. Although I only listened in on one day out of the four day conference, the research other universities were doing were astonishing. During the last week I continued culturing lungs in tissue culture, and observed the mechanical effects of Hepatocyte growth factor (HGF) and Transforming Growth Factor Beta 1 (TGFβ1) over the course of three days. I also learned 2D patterning from Sriram, who was really helpful throughout the process. I stamped 2 gels onto coverslips, and luckily one of the two came out alright. It was a great experience but it was definitely a lot harder than I could of imagined. The last week was essentially dedicated to compiling all my images and finalizing my research. I had a great time at the laboratory and really thank everyone at the lab for making me feel welcome. 

Week 8 - 10

Hello, this is Jacky Jiang from McAlpine group in Princeton. It has been a long time from last post. In the past three weeks, we keep on testing the efficiency of our methods to make thylakoid and the concentration of our chlorophyll.

To get better concentration, I need to come up with different methods of producing the thylakoid. As I have tried the classic procedures, the basic steps would be similar. First of all, I need to use a lab blender to blend the mixture of alginate leaf pieces with grinding buffer. After we get the solution, we will put it into the centrifuge for the pellet. The pellet we get need to go through another step, which is called resuspension. In this step, we put the pellet into the washing buffer and do the resuspension procedure. Then, we need to go through few more times of centrifuge to get the concentrated thylakoid. To make modification, I tried different kinds of centrifuge rate, which made the composition of the pellet different. This change could be very critical. Since the nuclei and other fragments of plant cells have different density, the centrifuge rate determines which component would be at the bottom of the pellet. What’s more, I also change the grinding buffer I used for blending the mixture. The different concentration of tricine would make the grinding level different, so that the size of membrane fragments would be different, too.

After I tried different methods, the results comes out that the classic steps with appropriate concentration of grinding buffer and high centrifuge rate worked the best. I also tried to change the sequence of centrifuging and buffer mixing, which didn’t turn out well in the end.

 To determine if our thylakoid is efficient enough, we still need to determine it by the concentration of chlorophyll. The experiment methods are the same as we did last time. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to 10 mL of 80% acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a 50 mL glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation.  

Our thylakoid concentration has improved a lot after we modify the methods. In the rest of the summer, we will move on to the electrical part of our project. 

Murphy Lab- Weeks 7-8

Hi, this is Richard, and I'm studying learning and memory at Princeton.  I will break up the last three weeks I spent at the Murphy Lab into two posts, and this is the first of the two.

I spent the majority of my seventh week repeating the egl-4::GFP nuclear localization assays that I had started earlier.  Since we don't really know what is supposed to happen in regards to the GFP-tagged EGL-4 protein entering the nucleus, I wasn't able to make too much sense of my results for my naive and trained worms.  In some cases, the GFP localized into the nucleus of the AWC neuron, thus causing the nucleus to flash a bright green, and in other cases, it was mostly the surrounding cytoplasm that was lit up.  Such ambiguity held true in both the naive and trained worms.  We did however, know what to expect with my adaptation assay, so we tried to confirm this result in which for adapted worms, EGL-4 enters the nucleus and for mock adapted worms, it doesn't.  However, while in some of the worms the EGL-4 protein had clearly localized into the nucleus, in other cases it seemed that it was present throughout the entire cell, not just the nucleus, as evidenced by the entire neuron being bright.  Unfortunately, I didn't have enough time to repeat this again and get a definitive result.

I tried my egl-4 and crh-1 cross again, and got to the point where I had candidates who had a 1/16 chance of being double mutants, but I did not have the time to run a PCR and isolate the successful candidates that I could use for my double mutant chemotaxis assay.  I did a PCR earlier, but none of the 30 candidates I picked out were identified as double mutants, and within a 9 week timeframe, it's pretty difficult to start from scratch and set up the same cross again.  I was really looking forward to this, because, as of know, according to wormbase.org, pretty much the bible of C. elegans research, no one has tested egl-4 crh-1 double mutants.  I could've been the first.  But not anymore.

Anyway, I spent the rest of the two weeks on a couple of STAMs and an LTAM as well, to help with Geneva's project.  The difference between an STAM and an LTAM (long term associative memory) is that rather than training the worms once (starve, food w/butanone, test), I must train them seven times in 30 minute intervals (starve, train, starve, train...).  I didn't test chemotaxis, but Geneva used the worms I trained to observe fluorescence.

These two weeks were definitely more eventful than some I've had in the past.

Tissue Morphdynamics Lab: Week 7-8

Hi, it's Danny again from Dr. Nelson's Tissue Morphodynamics Lab and its been about a week or so since my last update. Most of my time has largely been devoted to working on lung morphogenesis or essentially lung development, but also I got introduced to a new and exciting topic regarding divisional axes in tissue. Although the two topics don't necessary overlap in terms of research, the work I did the previous year at a UCLA Head and Neck Cancer Lab is very similar in terms of techniques but very different in terms of direction.

Sriram's project focuses on the effects of endogenous stress and motility in cell division. In previous experiments, it was shown that when two cells are attaches at opposite poles of another cell, they tend to create a polarity and divide along a parallel axis. Additionally, it is known that if there is no endogenous stress on the walls of a cell, the cell divides in an unpredictable manner. The objective of Sriram's graduate work is to determine how endogenous stress and cell motility affect cells' divisional pattern on 250 to 500 micron squares.

The process in create a small micron square and treat it with the necessary drugs and cells is a rather long but straightforward. Using soft lithography one can etch patterns into a film that can be used as a base for future experiments. Lithography is useful because it can create any pattern or shape but the pattern that is desired for this particular experiment is 250 micron to 500 micron squares. An essential material of the experiment is Polydimethylsiloxane (PDMS) masters, which is made by mixing cross-linkers to PDMS in a 1:10 ratio and vacuuming out an air bubbles. PDMS masters can then be applied to the lithography film which will result in a negative copy of the film. Then the negative PDMS masters stamp is coated with silane, a substance that prevents PDMS from sticking to itself. After the silane has dried, the PDMS masters negative can be used as a stamp and reused multiple times, until the quality of the stamps produced is subpar. PDMS masters is applied to a negative base and cultured in a thermo-regulator at about 60°C for 3 to 4 hours. As the PDMS masters is being cultured, one can spin down cover slips with PDMS masters on it. Once fours hours has passed, the positive copy is removed from the stamp, which is then cut into little squares using a razor. The PDMS masters squares are then treated with fibronectin protein and then stamped onto the PDMS cover slips. These cover slips are then treated with mammalian cells with a variety of drugs and analyzed using a con-focal microscope over 12 hours.

The machine used to spin down PDMS on cover slips
in order to create an even layer.

PDMS and cross-linkers (right)
Vacuum used to remove air bubbles (left)

I was supposed to attempt to make a cover slip treated with cells this week, but the fume hoods in the culture room were replaced due to ventilation issues. Sriram told me that the insides of new fume hoods are usually quite dirty and suggested that we wait some time before culturing and analyzing new cells. The past two weeks have been a mixture of chicken embryo dissections and imaging lungs as well as a lot of Imaris and image analysis.

PDMS masters stamps that will eventually be covered with fibronectin proteins.  

The next week is going to consist of micro-dissecting about 4 lungs in the morning and 4 lungs in the afternoon for day 4, day 5, day 6, and day 7 chicken lungs in order to start preparing for my poster and presentation. I will also hopefully attempt to go through the aforementioned process with the help of Sriram, if the fume hoods are restocked with equipment by then.

In terms of life outside of the lab, I've been working on my senior thesis paper over the summer and started on the dreaded summer homework. Josh has just left his lab, so if he reads this, hopefully he got back home safely. It's been a great 8 weeks so far at the lab and I've learned so much and met so many great people.

On a side note, today is Friday and I just got a new assignment to start going back through a set of data and count the number of cells in the data that has over 110 cells at around 11 hours. I've been counting cells for the past two hours because I would really dislike doing this on a Monday morning, so if you need anyone to count up to 150 in 2's as quickly as possible; I'm your guy. 

Week 5 to 7

Hi, this is Jacky and I am working in McAlpine Lab at Princeton. 

These three weeks have been very busy. Our projects have been moving to the first critical point, which is about measuring the quality of the thylakoids we make.

                The first test we perform is chlorophyll concentration measurement. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. Once the chlorophyll concentration is determined, the total chlorophyll yield should be determined by multiply the chlorophyll concentration in mg /mL times the volume (mL). Once the chlorophyll concentration and the total chlorophyll yield is known, the chlorophyll concentration should be adjusted by adding the appropriate amount of Washing Buffer or by centrifuging again the thylakoids and resuspending in the appropriate amount of Washing Buffer.

                By determining the concentration of chlorophyll we make, we can decide if the procedures we use is appropriate since there are a lot of different methods that we are able to choose from literature. It is always important in science research to try as much methods as possible.

                                          

                What’s more, we need to test the efficiency of the thylakoids we extract. In this case, we need to use a chemical method called Hill Reaction. As we learned in Biology class, during light reaction photosynthesis, electrons will end up in NADPH. In Hill reaction, we need to separate thylakoid with stroma so that NADP won’t be available. Instead, we will put DCPIP, a blue oxidant, into the solution. DCPIP is blue in its oxidized form, and becomes colorless when it is reduced during the Hill reaction. Thus, the rate at which electron transport occurs in the Hill reaction can be measured spectrophotometrically (at 620 nm) by following the change in absorbance of DCPIP as it accepts electrons from the electron transport chain. To perform the Hill reaction, a sample of a chloroplast suspension will be mixed with the Hill reaction buffer (containing DCPIP) and exposed to light for a series of 30 second intervals. After each exposure period, the absorbance of the DCPIP will be measured. The absorbance values can then be plotted versus time to determine the rate of DCPIP reduction as a measure of PET.

It turns out that the thylakoids we make are pretty efficient. We will keep on testing and trying for next couple days and shift our focus to other aspects. 

On to the Next Phase-- Weeks 4-6 at the Murphy Lab

Hi everyone, I'm Richard and I'll be talking about my past 3 weeks spent at the Murphy Lab, which examines various aspects of the aging process in C. elegans.

Week 3 was a definite struggle.  I ran 3 STAMs with my egl-4 mutants and wild type worms, all of which produced results opposite from what I expected, and since the entire lab was in California at the International C. elegans meeting, I was pretty much on my own when trying to figure out what exactly went wrong.  Geneva hypothesized that the worms were too young, so I made sure to bleach the egl-4 mutants a few hours earlier than I had previously done, and luckily, I was able to confirm my expected result in another STAM.  The rest of the week was spent primarily on preparation for future experiments and analyzing my data.

Week 5 was, for the most part, uneventful.  Originally I had planned to do PCR and run a gel to confirm that my egl-4 crh-1 mating worked to produce double mutants, but since a previous PCR and gel showed that I had no double mutants, I decided it would be best to re-do the original cross. So, once again, the week was spent mostly preparing for the next phase of my project, examining EGL-4::GFP nuclear localization.

For the nuclear localization assay, I first starved my egl-4::GFP worms (these are not the same as egl-4 mutants; rather, they are wild type worms with the EGL-4 protein tagged with GFP so that fluorescence can be observed).  I waited an hour, as I usually do for my chemotaxis assays, and then put the worms on conditioning plates spotted with butanone.  After another hour, I transfered these worms onto a hold plate and prepared microscope slides as quickly as possible to ensure the worms didn't lose their food-odorant association, and then examined fluorescence under a microscope.  To do this, I first locate the head of the worm, at about 60x magnification, and then switch to the option that allows me to view the RFP tagged (red color) AWC neuron.  At this point, the entire neuron, except for the nucleus in the center, flashes a bright red.  I then switch to the option that allows me to view GFP (green color), and depending on whether the nucleus is a bright green or still dark, as observed under RFP, I can tell whether or not the EGL-4 protein has entered the nucleus. I repeated this procedure with naive (untrained) worms, and also with adapted worms (adaptation is when a long, repeated exposure to a certain odor actually diminishes the worms' response to that same odor).  Today I will be analyzing the images I took during these nuclear localization assays.

Overall, I've enjoyed my experience in lab.  However, my days are not always as busy as one would expect, and I've had a couple of setbacks with various aspects of my project, which have consequently resulted in less eventful days.  I guess failure is something every scientist has to deal with sooner or later.

Tissue Morphodynamics Lab: Week 6

So this is final addition to my three part post. The sixth week was just as exciting and new as the fourth week, as I was given a new project to work on for the rest of my stay. Needless to say, I had to do a little bit more reading of reviews and articles to understand the basics of cell mechanobiology, but it was all very similar to the reading I did the previous summer at a Head and Neck Cancer Lab at UCLA. The post-doc, Jason P. Gleghorn, and the graduate student, Sriram Manivannan, explained to me their hypothesis, their prediction, and their results so far regarding cellular motility and cellular divisions along an axis. It was essentially the same topic I listened in a group meeting two weeks before.

The specific aim of their research is to understand the mechanics behind cellular division and how rotational axes and endogenous stress affects the division of cells.

In addition to culturing, dissecting, fixing, imaging, and analyzing chicken lungs, I was given the task to analyze their image data of hundreds of 250 micron by 250 micron squares of cells over a 12 hours period in 6 minute intervals. There are 121 images per time lapse that are compile into a TIF file and then analyzed using Imaris, a cell tracking program. Then using MatLab, the data of all the positions of the parent and daughter cells are compiled into an image that shows the overall positions of all the time lapses.

Imaris program. Tracking the parent cell. 

Imaris program, Tracking the two daughter cells. 

Jason usually deals with compiling the data, whereas Sriram used to analyze the images as well as create 2D patterns, culture the cells, and produce the images on the confocal microscope over the weekends. Now that I'm analyzing the majority of the images, he has more time to write up his paper regarding cell motility as well as prepare for group meetings and conferences.

In addition to analyzing the image data, I am also learning 2D patterning, micro-fabrication of PDMS masters using lithography, and culturing mammalian cells in vitro. It is all very interesting also a flood of information. For the moment I am learning the basic steps and making sure that I understand the procedure before I attempt to make a pattern by myself.

Over this past week I have looked at about 60 or so time lapses of cells on 250 by 250 micron squares. I have just finished the control, and after analyzing the data in MatLab, the results shows a shape that closely resembles that of a square with rounded edges. The data is all very interesting and it is exciting to analyze the data as no one knows what the results will be or how different drugs that affect endogenous stress will affect the overall patterning of the divisions.

Tissue Morphdynamics Lab: Week 5

So, its Danny, again, and this post will hopefully be a lot shorter than the last one. The fifth week was when the multiple hours in the afternoon spent dissecting chicken embryos would start to pay dividends in research. At the start of the fifth week I started to culture lungs in tissue culture, which was very challenging due to time constraints. In the previous post I mentioned that lungs would only be preserved in PBS solution for up to an hour, and then after an hours they would be considered "dead". Therefore I only had an hour to dissect four or more day 5 chicken embryos, extract the lungs and place them in culture dishes. Despite having had numerous hours of practice, I never felt pressured in dissecting the embryos as I had all afternoon to practice.

Needless to say, my first attempt at dissecting chicken embryos was mediocre at best, as I managed to get three chicken embryonic lungs in a little under an hour. Then Amira, the research specialist, showed me how to culture the lungs in wells. Additionally, all the work was done in a chemical fume hood and all surfaces, bottles of media|serum and gloves were sprayed down with 70% ethanol in order to prevent contamination.

To make the media that the lungs would grow in, we added 10mL of Hyclone DMEM/F-12 (1:1) glutatmine and 500µL of FBS serum into a 15mL sleeve tube. In order to mix the solutions, we simply turned the sleeve upside down multiple times, instead of vortexing it. We then got a 6 well plate and added 2mL of FBS|DMEM solution into a well using a 5mL pipette. Next we placed a single Whatman Nucleopore Track-Etch Membrane into the well with forceps, with the glossy side of the membrane facing down towards the bottom of the well. The Nucleopore Track-Etch membranes ensure no contamination, have a smooth flat surface that allows for high visibility of particles during a microscope, and have high chemical resistance. The membrane essentially sits suspended in the media. Lastly we used a transfer pipette to move the three lungs from the PBS solution on the membrane. The lungs are then grown in a carbon dioxide incubator that cultures the epithelial cells. We take pictures of all three lungs at 0 hrs, 24 hrs, and 48 hrs, which will later be used for analysis.

Day 5 Lungs (0 hrs)

Day 6 Lungs (24 hrs) 

Day 7 Lungs (45 hrs)

The purpose of growing the lungs in tissue culture is to analyze the results using ImageJ, a program that analyzes the area, perimeter, and other useful measurements of images. This data is then compiled onto an excel sheet, which is in turn used to create a graph that can be used to determine the tendencies of lung development. When the lungs are further treated with different concentrations of lungs, these graph also have varied results. The following are images of both the analysis I have done so far as well as the work that the lab has already done and published in an journal. In the excel sheet graph, the x axis represents the area of the epithelium, whereas the y axis represents the number buds located on the epithelium. The results also include all date from 0hrs to 48hrs.

Lung Development Graphs
Source: Jason P. Gleghorn et al (2012)

Lung Development from a few samples.

At the end of the fifth week, the microscopes were finally available and we were able to image the lungs I had stained using a phase contrast microscope. The confocal and phase contrast microscopes at the lab were to be used with extreme caution, as they had broken down multiple times over the past two months, according to Amira as well as other post-docs in the lab. Therefore I watched Amira image the lungs using HC-Image Live, and found it very interesting. Using a transfer pipette, we placed the lungs with PBS solution onto glass cover-slips and then using a beam of light, we found the optimal wavelength (around 460 nano-meters) that produced a good quality image. It was really interesting to see how the microscope was used, as there was a lever on the side of the microscope that changed the frequency of the light. The images are in black and white, but in the live feed from the computer they were a nice turquoise.

Day 5 Embryonic Chicken Lungs

Day 7 Embryonic Chicken Lungs

Tissue Morphodynamics Laboratory: Weeks 4

Hi, this is Danny with a couple of updates regarding my progress in Dr. Nelson's Tissue Morphodynamics Lab. My next three posts are going to be a load of information, as I learned a lot of new material and methods over the course of past three weeks.  I understand that these posts are little late, but it could not be helped, as my laptop with all images crashed. I felt that the words I was writing would not do the  techniques or methods any justice so I figured I would wait for my laptop and my images. Just to note, these three posts are actually just one post, but I just prefer to have the weeks organized as there is going to be a lot information being posted.

My lab bench where I stain, aspirate and fix the lungs.

One of the three dissecting microscopes I use.  

The fourth week at my lab was definitely more exciting and productive than previous weeks, as there were no interruptions or unforeseen obstacles. In addition to a new high school student, Chris, from Montgomery High School joining the laboratory, there were also three four new senior thesis students and an undergraduate student. Needless to say the lab got crowded very quickly and I found myself changing desks numerous times. Although the dissecting microscopes were usually occupied during the mornings, I found myself with microdissecting chicken embryos the entire afternoon from mid-day to around five. My first day of microdissecting day six chicken embryos was somewhat embarrassing, as I microdissected ten or so chicken kidneys instead of the lungs. Having been told that the lungs are slightly larger in day six embryos than day five, I found myself with a plate of chicken kidneys. Although ten or so chicken embryo unfortunately were lost, I found that microdissecting chicken lungs was a lot easier than trying to take out the kidneys. Around the middle of the week I already had fifteen or so day 5, day 6, and day 7 chicken embryo lungs waiting to be fixed, stained, and imaged. 

Although I went into a little detail regarding the staining of the epithelium in my previous post, I figured that I would go into a little bit more detail, as I see other EXP students are staining as well.

Throughout the microdissections and the staining procedures, Phosphate Buffered Saline (PBS) is crucial in order to preserve the lungs from an hour up to months. After successfully dissecting at least six chicken embryo lungs I head over to my lab bench with my dish of lungs submerged in PBS and start to fix the lungs. In order to make the formaldehyde solution for fixation, I add 10mL of stock 16% paraformaldehyde (PFA) solution to 30mL of 1x PBS, resulting in a 4% PFA|PBS solution. Then I add the lungs into a well, usually three or four to a well, and then aspirate the excess PBS solution using an aspirator and add the 4% PFA|PBS solution to the culture dish. I then place the dish on a shaker for amount 15 minutes and then aspirate the PFA|PBS solution and washing the excess PFA|PBS solution three times with PBS. Next, I prepare a Phosphate buffered Saline with Triton X solution (PBST) by adding 1.5mL of Triton X into 500mL of PBS, and add a stir bar and leave it on a stir plate for about 30 minutes in order to make a 0.3% PBST solution. Triton X is a very viscous solution and is used in order to make holes in the tissue and mescheyme, which allows for antibodies to react with the epitopes for primary binding in staining. To continue fixing the lungs I add the 0.3% PBST solution to lungs for about 15 mintues on a shaker. Then I add a blocking buffer, which consists of 4mL of 10% goat serum (GS) and 30mL of 0.3 PBST, in order reduce background noise when staining and imaging. I leave the lungs submerged in in the blocking buffer in 4°C for four hours on the shaker, as GS tends to attract bacteria if left at room temperature. After four hours I add the primary antibody, which consists of 1mL of PBST and 5µL of LCAM. After four hours and aspirating the blocking buffer, I add the LCAM primary antibodies overnight at 4°C on the shaker. When I return the next day, I aspirate the LCAM solution and then wash the lungs in PBST for about 4 hours, aspirator and adding sterile PBST every hour. Then I add the secondary antibody, which consists of 488 nano-meter (turquoise color) goat-a-mouse serum. I wrap the plate of wells in tinfoil and leave it overnight. When I return I usually spend up to two days constantly aspirating and adding new PBST to the well of lungs, in order to prepare for imaging. As one can see staining takes about half week at a time, so it is best to have many lungs to stain at once than repeat the process multiple times. 

Although the fourth week mainly consisted of microdissecting, messing up microdissections, and then staining the lungs in order to prepare for imaging the following week. I also learned a lot more about the work others were doing in lab through the group meetings, which consisted of Oscillatory Rotation and Cell Motility and the Mechanics in Meschyme-free branching. What was interesting in the latter topic was that the post-doc cut the distal tips of chicken embryos, which reportedly are places of high stress and proliferation, and embed them in matrigel which was submerged in fibroblast growth factor (FGF), a family of growth factors that are key in embryo develop. Using time lapse imaging on the confocal microscope, he was able to create a animation of the terminal end buds actually growing out of these square matrigels in culture. Although similar works has been done, which I will provide an image in a second, I don't believe anyone has necessary done the work recently using modern technology. 

An  general idea of the project discussed during the group meeting.
Source: Fibroblast Growth Factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung

Lastly, in between staining and microdissecting chicken lungs, I was busy learning other useful techinques that may or may be useful in the near future. I learned the process behind making complementary DNA (cDNA) and quantifiable polymerase chain reactions (qPCR), which is similar to the polymerase chain reactions (PCR) we did in AP biology. In short, the entire cDNA process consisted of following a instruction booklet sent from Thermoscientific Fisher and numerous attempts to find a optimal amounts of solutions to add. qPCR was similar to PCR except that the procedure would allow for one to quantify the relative amounts of increase in DNA overtime relative to other samples using spectrometer, whereas in PCR one could only analyze the amounts of DNA after running the DNA through gels and imaging them in ultraviolet light. 

The fourth week was a lot of new and exciting research, as well as new and exciting methods and techniques. I learned a lot during the fourth week and got to know a lot of the people in lab, besides Amira and Ben. 

Week 3-4 in Princeton

My name is Jacky Ziwen Jiang and I am working in McAlping lab at Princeton for this summer. It has been four weeks and our research has been going pretty well.

As the first two weeks were mainly about planning and chemicals ordering, we turned in to the actual working mode the past two weeks.

The main material we need to prepare is thylakoid, the main factory where photosynthesis takes place. To get thylakoid, the method is not that simple as just cut leaves into small pieces. First of all, we need to choose the right plant leaves for the thylakoid extraction. After reading certain amount of articles, spinach leaves has the relative high concentration of chloroplasts in their leaves, which we will choose for our experiments. After we bought the spinach, there are two more buffers we need to make for the thylakoid extraction. One is grinding buffer and the other one is washing buffer.

The whole process is filled with multiple complex procedures. First of all, the precise measurement of different chemicals we need takes consistent attention without vacillation. After that, I need to use a lab blender to blend the mixture of alginate leaf pieces with grinding buffer. After we get the solution, we will put it into the centrifuge for the pellet. During this process, I also learn new knowledge of the unit conversion between rpm and g, which show the rotation strength. The pellet we get need to go through another step, which is called resuspension. In this step, we put the pellet into the washing buffer and do the resuspension procedure. Then, we need to go through few more times of centrifuge to get the concentrated thylakoid.

The solution looks clearly green and fresh. These two weeks’ experiments give me a great learning opportunity of thylakoid, which gives me a further understanding of the knowledge I have learned in class. This is a great example of practice the knowledge I learned in actual experiments.  

STAMs-- weeks 2&3 at the Murphy lab

Richard here, studying learning and memory in C. elegans in Dr. Murphy's lab at Princeton.

For the past two weeks, the primary focus of my research has been to perform short term associative memory training (STAMs) on wild type (N2) and egl-4(ky95) worms.  I may have given a brief description of STAMs in an earlier post, but I'll go into more detail this time:

The experiment starts off with several plates of a strain of worms, which have been bleached so that the worms being tested are all about the same age.  Using M9 buffer, I wash the worms off one of the plates to serve as my naive testing group-- I examine the worms' responsiveness to the chemical butanone without any prior conditioning which will enhance their response.  Then, I wash the rest of the worms off the plates and into a 15 mL tube, where they starve for about an hour.  When this hour has passed, I transfer the worms onto several conditioning plates, with food, and spot a small amount of butanone on each plate so that after an hour of conditioning, the worms will have a developed a strong association between food and butanone.    To test this association, I prepare chemotaxis plates, which look like this:

Chemotaxis assay of wild type right after conditioning-- all the worms are attracted to butanone (left)

The dots on the right and left are both spotted with sodium azide, which stops the worms from moving, and the right and left dots are spotted with ethanol and butanone, respectively.  I put about 100-300 worms on the dot at the bottom, called the origin, and after an hour, I take a picture of the plate and use a digital sorting program to count the worms.  This entire process is called a chemotaxis assay, and I need to perform one at various time points: right after conditioning (0 hr), 30 minutes after conditioning, 1 hr, 2 hr, 4 hr, and 6 hr, using three chemotaxis plates per time point per strain in order to ensure accuracy. The worms being tested at later time points are placed onto hold plates which contain food but no butanone. Factoring in the two hours it takes to starve and then train the worms, the entire experiment lasts 8 hours.  However, there are large chunks of time in between, which gives me time to perform other preparations needed for future STAMs, and time to just relax.

My second week, I performed three STAMs with just the wild type strain, to get acquainted.  My third week, I performed three STAMs with both the wild type strain and the egl-4 strain.  It is known that the egl-4 strain retains its association between food and butanone for several hours, unlike wild type, which loses this association almost completely 2 hrs after conditioning, but during all three of the experiments I performed last week, the egl-4 strain actually displayed even worse chemotaxis towards butanone than did wild type, which is the exact opposite of what was expected.  Two possible explanations immediately come to mind: 1) I am a genius who has just proven the scientific world wrong, or 2) I'm an idiot who, despite having performed the same exact experiment three times, still managed to completely screw up.  Option number 1 sounds  flattering, but is highly unlikely.  Option number 2 seems more realistic, but while there are many things at which I am inept, I am certainly no idiot, never have been, and I am 150% sure that I performed these experiments with as impeccable timing as I could achieve.  With this being said, at this point, I'm not sure what exactly went wrong, but now that my grad student, Geneva, along with the rest of the lab, is back from the International worm meeting at UCLA, hopefully we can figure something out.

In the meantime, I was able to cross my egl-4 mutants with my crh-1 mutants to produce heterozygotes, and then allow those to self fertilize to produce possible double mutants.  In order to verify if any of the offspring chosen are indeed double mutants, I performed PCR and then ran a gel.  However, if there are serious problems with my egl-4 strain, as evidenced by my extremely odd results from my egl-4 STAMs, then this pursuit may be in jeopardy.  All I can do is hope for the best.

Tissue Morphodynamics Labratory: Weeks 2-3

Day 5 Chicken Embryo

My second week of the lab was a little chaotic due to the Princeton bomb scare, which set back  my laboratory safety training which in turn set back my general laboratory training in microdissections. Due to the Princeton bomb scare, I was unable to start microdissections because the chicken eggs had grown past a viable embryonic stage for tissue culture. My laboratory manager told me it would be another week before I would able to start dissections for tissue culture. The second week therefore consisted of more reading and more articles about different topics, such as RhoA pathways and e-cadherins. Through these articles I learned about how pathways regulate tissue in the epithelium as well as the importance of e-cadherins in developmental biology. At the end of the second week I was also given a host of data and images to organize, analyze, and graph.

Lungs of Day 5 Chicken Embryo

At the start of the third week I finally started to learn the multiple techniques and solutions I would need to use in order to analyze chicken lungs under a confocal microscope; however the embryos were still unable to be used for tissue culture. I started by dissecting the lungs out of a day seven chicken lung under a dissecting microscope using two 5mm forceps. In order to extract the embryo from the egg, I took a syringe and poked a hole at the bottom of the chicken egg and removed some fluid in order to lower the yolk height in the egg. Then using a scissor, I cut a semicircle at the top of the egg, removed the shell and scooped the chicken embryo out of the egg and on to a petri dish filled with saline PBS solution. After successfully dissecting four lungs out of chicken embryos I learned how to fix the lungs in preparation of staining. Fixing the lungs is essentially using a solution in order to preserve the tissue of the organ and allow for storing overnight. After fixing, I stained the lungs using LCAM antibodies, which are antibodies that bind to e-cadherins on the epithelium and creates 
contrast between the epithelium and the mesenchymal tissue.  

 

Fixing and Staining of 4 Day 7 Chicken Lungs

Towards the end of the third week I was given more data to analyze, which was mainly measuring the lengths and area of the epithelium using Adobe Photoshop and ImageJ as well as counting the number of terminal end buds of previously stained chicken lungs. I also attended a microfabrications seminar with others in my lab in order to better understand how to amplify signals using biosensors on a plate. 

New Project about plant

As we have finished our 3D printed bionic ear, our group is starting a new project. We are basically preparing for the new one, which will be about plant and energy harvesting.

In this project, I need have more reading since I need to contribute a lot to the experiments design and performing, which includes the material choosing and methods design. As I am pretty familiar with the knowledge of plant photosynthesis (thanks to my great AP Biology class), I will be more specifically in charge of the mechanics part of the leaf, while I will learn more about the electronic part.

What’s more, instead of alginate and bovine cells, we will use totally different material for this time, which means we need to design new setting for our lab. These days, through the information we have, we are deciding the material and other electrical components of the project, while the material needs to be reconsidered. We are also preparing for the interview from MIT’s Technology Review, which records the whole process of our 3D printed bionic ear. So a new fresh ear is coming.

One of the most exciting things taking place these past two weeks is the presentation of the new project in our group meeting. This is the presentation that I have longest talk about our idea, in which I explain how our research would go. In the presentation, I first mentioned the necessity of plant energy harvesting and the general information about photosynthesis including photosystem and electron transaction way. Later, I talked about the idea of combining nanotechnology and plant biology, and the specific methods that we were going to follow. The presentation went well.

In the next week, we will start the new project and I cannot wait for the experiments with plant.

Worms, Bombs, and Everything in Between

My first day was a bit of a surprise.  After extensively studying and ultimately writing my review on the insulin-like growth factor 1 (IIS) pathway and its roles in aging in C.elegans (a nematode), I learned that the project which I would be undertaking had nothing to do with the pathway.  But I'm actually glad that Dr. Murphy pushed me in a different direction.  Before coming to the lab, I couldn't really decide which of the various components of the aging process, including reproductive aging, oxidative stress, and learning and memory, I wanted to focus on, so I simply said that I wanted to do something on general longevity regulation, which, in hindsight, was probably the least attractive of my options.  I was assigned to work with a grad student, Geneva, who focused specifically on learning and memory.  We came up with a project in which I would train wild type, egl-4 (which functions in the AWC neurons), and egl-4 crh-1 double mutant worms to develop an association between a specific odor and food, and then examine if such an association is still present after a certain amount of time.  Unlike the IIS pathway, which is so well documented, the pathway in which EGL-4 acts is not, so it'll be really cool to study something which isn't as well understood.  And since I'll be working for 9 weeks, I might get to make more double mutant strains to test.

So basically, the rest of that first day consisted of some basic training of techniques, i.e. how to bleach the worms to obtain the eggs (which sucks for the worms because their bodies get completely dissolved), seeding plates (putting bacteria on them); a 2 hr long lab meeting, during which I was hopelessly lost; and reading 2 articles.

Tuesday was weird, to say the least.  Geneva, who is six months pregnant, needed to get some sort of test from a doctor, so she wasn't coming in until 12.  I arrived at 8:30 as always, and got cracking on reading those last 2 articles I was supposed to.  Some time between 10 and 11, I overheard two people in the lab I was in (not the Murphy lab because Geneva's office is in another lab) mention a bomb threat.  I thought this was simply a drill, so I just sat at my desk and turned to Gunnar, a postdoc in Dr. Murphy's lab who, like Geneva, has his office outside the lab, asking what to do.  He wasn't sure either, but a lady told us all to get out, so we did.  It wasn't a chaotic scene, however.  There was no sense of panic among everyone, and once we got outside we saw a mass of people walking towards the parking lots.  Thank God Gunnar was there, because he was able to drive me home.  Otherwise I would've been wandering around like an idiot.

Wednesday wasn't too eventful.  I learned how to 'chunk' worms (cut out a piece of agar on a plate of worms and transfer it to another), but other than that, I didn't have much else to do in the lab, so I mainly sat in Geneva's office reading articles and learning more about C. elegans.  In the afternoon Geneva and I went to another building, because she needed, for her project, to use a biosorter (a very fancy, intricate machine) to separate her worms which fluoresced under UV light (due to expression of GFP) from those which did not.  There aren't too many low points when it comes to research, but this has to count as one of them.  We essentially sat there doing nothing for three hours just waiting for the machine to count the tens of thousands of worms she had in her samples. And by the time I left about three hours later, the process still wasn't over.

The next day I attended safety training and I learned how to pick up individual worms, by using an extremely thin 'spatula' to scoop the worms up while I observed them under the microscope (these things are only about 1 mm long).   At first I couldn't pick up a single one as I didn't want to puncture the agar on which the worms grew, but after a few minutes something just clicked and I was able to pick them up with ease.  The funny thing is, on Friday, after I picked up the worms, they wouldn't get off the spatula, so I tried to scrape them off, but when I did that I would get agar stuck onto the spatula, which made it even harder for the worm to get off. There were several instances in which, after approval from Geneva, and a bit of guilt, I stuck the spatula into a gas flame and burned the worm to a crisp. Yes, I feel terrible about doing this, but I really had no other choice.

The rest of Friday was spent preparing for my first STAM (short term associative memory training) on Monday with wild type worms.  I needed to bleach my worms (dissolve their bodies; keep the eggs to make sure all the worms I use are the same age), and seed my plates with E. coli.  I also set up a mating of an egl-4 hermaphrodite and a crh-1 male so that I get heterozygotes in the next generation, from which I can ultimately select double homozygous recessive mutants.

So far I've enjoyed my time in the Murphy Lab.  Next week will be busy, as I need to run three STAMs and  do the necessary preparation for each (bleach worms, seed plates, chunk plates), but I'm looking forward to it.  If there's one thing I'd say I don't like about the lab, it's that I have to work extensively with E. coli, and that stuff just smells terrible.

Tissue Morphodynamics Labratory: Week 1

The first day at my lab was largely uneventful and somewhat stressful at first. Jacky's lab didn't start until 10, so he was kind enough to help me find my lab. We went to the Engineering Quad assuming that my P.I. was still working there, but was told that she moved to Hoyt Laboratory. One man at the Engineering Quad kindly directed Jacky and me towards Hoyt and said that it was relatively close to Frick's Chemistry Lab. As we walked towards the general direction of Hoyt, we asked a man walking out of Frick's if he knew where Hoyt was. After some deliberation, he pointed us towards the "new chemistry lab," which ended up being the wrong place. Then a man coming out of the new lab pointed us back towards Frick's. Eventually we ended up looking up Hoyt on our phones and realized it was about a good twenty steps from where we asked the first gentleman for directions. That adventure lasted for about 30 minutes before I arrived at the lab.

The rest of the day was spent reading articles and reviews from past graduate students, postdoctoral fellows, and other labs in order to further understand tissue development in chicken and murine lungs. I met the research specialist that would be teaching me the multiple techniques, Amira, as well as an undergraduate student, Ben. Both were very welcoming the first day, although they clearly had a lot of work to do. The following day started off with more papers to read but ended with a demonstration of chicken embryo dissection. Although I wasn’t able to dissect or extract the chicken embryo, it was nonetheless interesting to watch Amira microdissect the chicken embryo with such ease and extract a chicken lung from the mess of tissue. The next three days were filled with more articles and I got through about 10 articles, 5 reviews, 1 graduate student’s dissertation, as well as Turing's Chemical Overview of Morphogenesis over the course of the week. The last one took almost two days because its complex math and formulas.  Next week, after I receive my lab safety training, I will start practicing dissections in order to competently prepare chicken lungs for experiments.