Teamwork Makes the Dream Work: Seeing the Results

Hey readers!

This week has been probably filled with the most ups and downs I've ever experienced... as you read last week, I started the week despairing about a complication at ASU: I could not run human blood samples in the particle accelerator. And I was getting frustrated.

Early this week, I felt as unprepared as Dunder Mifflin for a Dwight fire drill.

However, I got a change in perspective... I owe a big thanks to Mrs. Haag, Dr. Herbots, and a few fellow interns like Grady Day and Ryan van Haren. I had been going into the lab daily, but I was just trying to accomplish tasks, and I would get frustrated if they didn't get done. I had forgotten what really mattered -- that I was doing my own experiment, and that even though things didn't go according to plan, I was lucky in a lot of respects. So, I pivoted and pushed harder to collect data and,  of course, learn some amazing stuff about HemaDrop™and biomedical engineering. Now, I feel like my research is finally hitting its stride.

Let's goooooooo

So, I'll take you through the work it took this week to get us there -- and then go in to what my Results section should look like.

Without human blood, we focused on using model liquids (like balanced saline solution and canine blood) to take our RBS measurements and then characterizing how human blood, saline, and canine blood dried on our samples. Armed with this new focus and new equipment (mechanical pipettes which deliver controlled volumes of liquid as small as 5 microliters), I am in a groove.

You have NO IDEA how much joy these pipettes brought me... I had been struggling with bad syringes for so long.
You can see my apparatus here, where I apply saline to the sample and then observe its drying with the microscope (we have videos!!).

I got to finish my 3LCAA analysis and make super super exciting progress on calculating uniformity. I even planned out an outline of my methods section with Dr. Herbots and Mrs. Haag, so let's talk about what I want in my results section.

I looked at some sources in my discipline to find some key qualities of results sections. Here are the sources I decided to investigate:

1. Acharya, Ajjya et al. “HemoClear: A New Thin Fluid Film Device to Control Blood Clot Formation.” American Physical Society Fall Meeting - Four Corners. 59, (2014).
   (Biomedical engineering thesis at Barrett Honors College of a fellow intern from the Herbots lab. This results section is very useful, as Ajju Acharya discusses the importance of a new technology called HemoClear, uses spectroscopy to measure elemental composition and qualitative observations to compare samples. Used mixed methods)
2. Depciuch, Joanna et al. “Phospholipid-Protein Balance in Affective Disorders: Analysis of Human Blood Serum Using Raman and FTIR Spectroscopy. A Pilot Study.” Journal of Pharmaceutical and Biomedical Analysis. 131 (2016): 287–296.
   (Paper involving the analysis of blood with spectroscopic techniques similar to RBS. Useful, as it shows the best way of portraying Rutherford Backscattering Spectrometry Spectrometry -- which I have to do. Purely quantitative)
3. Thomas, A. et al. "On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed- phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites." Journal of Separation Science 33, 873 (2010). (Paper discusses the benefits of a new technology for analyzing blood spots with liquid chromatography... presents specific chromatograms with detailed figures including models of the samples. Employed mixed methods.)
4. RELATIVELY HEALTHY 17 YEAR OLD INDIAN MALE et al. “Electrolyte Detection by Ion Beam Analysis, in Continuous Glucose Sensors and in Microliters of Blood Using a Homogeneous Thin Solid Film of Blood, HemaDrop™.” MRS Advances (2016): 1–7.(Paper proposes a new technology and tries to demonstrate uniformity of samples with RBS and PIXE. Purely quantitative)

I picked these sources because they were all in the field of biomedical research (more specifically, biomedical engineering or applied physics), just like my research. Before you start to think that I am that worst kind of arrogant jerk who cites his own papers (which in fact I am), I added #4 as a results section I could destroy because I know the mistakes I made in that paper -- mistakes which I am not keen on repeating, as they would make my results section not understandable in my AP Research paper. So without further ado, here's what I found:

#1 began with a restating of the purpose and what methods were employed to answer the major research questions. By doing this, Acharya clearly showed what he sought to find out, and then explained how he got the results from the methods he employed.

#3 and #2 showed very clear figures with full explanations, so that the figures basically could stand alone. From my Cancer Bio class with Dr. Scaling, I have found that figures are a really really important part of biomedical research papers, as many in the field are familiar with methods you are using, but want to see the results for themselves. #4 does label parts of the RBS Spectra pretty well, but the figures do not stand alone and little quantitative data is shown apart from the graph. To improve upon what I did in that previous paper, I have devised a more rigorous method of calculation of uniformity apart from just comparing spectra visually -- the subtraction method. With this, I can show a bar graph along with a spectra (scatterplot), which will clearly show elemental composition and error levels. Relating back to error is extremely important, as the impetus of the project started with comparing HemaDrop to previous blood tests and even Theranos.

#2 did a fantastic job restating data from the tables/figures in different ways that allowed for a greater understanding of the paper and "planted the seeds" for its conclusion section. By showing data in percentages (i.e., for me error values for measurements), graphs, and raw values, #3 made sure the readers understood the meaning of the results. #4 was lacking these explanations, since I kind of just spilled out a bunch of numbers and calculations without a lot of explanation, which made the paper esoteric and convoluted.

However, both #4 and #5 did a nice job of showing pictures of the samples along with their quantitative data. This practice really made the plots more digestible. #1 had nice graphical displays with keys on how to read the graphs and why the axes/scale were chosen. Again, the figures and explanations in my paper were lacking.

#1, Acharya's thesis, provides a really interesting model for me to present my qualitative results, as he used a mixed study as well. He separated the qualitative data from the quantitative data a lot though, so I think using the qualitative characteristics as signs of quantitative lack of uniformity will allow me to put those sections of my results in better conversation for my conclusion. He used tables very effectively for observations, which I plan on doing.

One element we discussed in class, which no one used were clear examples of specific qualities (for qualitative) or calculations (for quantitative). Space permitting, I think showing an illustrative example of calculations and images will really allow readers to understand my paper better.

Phew -- that was a lot. So, from that I think I will have 3 parts of my results section. (1) 3LCAA data which characterizes the hydrophilicity of the samples, (2) Qualitative coding data, and (3) Quantitative uniformity calculations. Can't wait to show y'all what I have next week!!

So, that's it for this week. I'll leave you with a sick time lapse gif of blood drying that I took last week in video form with a microscope. You can kind of see the film sucking up the water -- which was our initial hypothesis!! HYPER-HYDROPHILICITY WORKS?!?! Stay tuned for more information next week!

20 min video sped up 10000% into a 12 second gif

Cheers,

Yash

P.S. I'd be remiss if I didn't thank all my fantastic fellow interns (old and new) who've been helping me out with my project: Grady Day, Saaketh Narayan, Jack Day, Ryan van Haren. You guys are amazing!

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The Obstacle Course: (Collecting and) Analyzing Data

Hey readers!

This week, I've been working hard collecting in the lab and analyzing data at night to try to finish my data collection on time. However, it seems like every bit of progress is met with a new obstacle to overcome, but that's the fun of it and the nature of the beast, I guess...

I'm the mouse, and data is my cheese.

So, without further ado, here's what I've been up to this week:

Monday, I (re-)set up the apparatus for 3LCAA, which is a technique I am using to characterize the hydrophilicity of the samples. I came in last Saturday to set this up, but someone ignored my notice and moved the camera which ruined fine-focus I had calibrated (you need HIGH HIGH HIGH resolution pics for these drops).

My reaction when I saw the camera after it was moved.

Once it was calibrated, I analyzed 3 of my 10 samples by applying drops of water, glycerine, and alpha-bromo. I finished characterizing all of the samples by Tuesday, so that was right on schedule.

Example 3LCAA image... look at how nice those drops are! We care about the angle each drop makes with the surface.

Since I knew that my data would not be complete by Feb 19th, Mrs. Haag generously granted me a 1 week extension. However, I still did not want to be behind, so I have been analyzing 3LCAA images at night to get those elusive contact angles and thus surface energies. This involved fitting an ellipse to each drop (~7 per sample with 3 identical samples and 10 of each type of sample) -- we can find the contact angle from the arctan of the y-offset and major axis (if you're interested, you can read the step-by-step directions in our lab's 3LCAA SOP written by my wonderful fellow intern Ashley).

Here's a screenshot of me analyzing some drops on Inkscape. Check out the ellipses!

I finished parsing the 3LCAA images, so now I only need to enter the contact angle values from my Inkscape SVG files (as shown above) into my master spreadsheet, which can calculate the surface energies. Once I have these surface energies, I plan to plot them by sample first on a bar graph, as shown below.

Here's an example of how previous students in my research group have plotted surface energies from 3LCAA

The advantage of this method is that we can see the contributions from the individual components to the total surface energy (the lower dots that sum up to the top dot). After showing this bar graph, I hope to quantitatively plot uniformity as the dependent variable responding to surface energy. In this scatterplot, each data point with coordinates (surface energy, uniformity value) would represent a sample (combo of coating and substrate). 

About uniformity...  now, it's obstacle time. ASU has been very difficult (basically stopping our research) because of our use of human blood, even though we had previously gotten approval from them. So, we have been working with them to allow RBS of human blood (where we get elemental composition and thus uniformity from) and qualitative observations with human blood. 

Me when the lab technician at ASU stopped us from running blood samples

But, there's not stopping -- we just gotta adapt. 

No, seriously, we can adapt... no excuses!

I decided to pivot slightly and prepare samples from saline and canine blood, since we are apparently allowed to run RBS and qualitative analysis on those types of samples. Although human blood is preferrable, both types of liquids are models for human blood (ions in a aqueous, or water-based, solution) and can also give us measures of uniformity. At the same time, I'd really like to do work with human blood, so we hustled and submitted a request to Environmental/Health Safety. 

So, right now, I have 1 day of RBS, and we are proceeding with RBS tomorrow, as planned, except on samples with saline and/or canine blood instead. I held off on pursuing qualitative observations because I may still potentially be able to use a lancet to apply human blood to the substrates, even if I can't analyze its elemental composition. I think it will still be a robust data-set, but please keep your fingers crossed that we hear back soon about using human blood, as planned. 

One "benefit" of this could be that I may have to alter my methods section to reduce the sections on sample collection, which could reduce my word count. But, as a researcher who wants his experiment to be the best it can be, I definitely do not consider that ideal. Whatever RBS data I get, I will obtain the elemental composition at 2 spots for each sample, subtract the values, take the absolute value, divide by the average, and find the mean -- this will give the standard error for each sample. To be implementable, it must be less than 10%. 

Here's an example spreadsheet from the control (no HemaDrop-coating).
I got errors above 30% (expected), as you can hopefully see.
The font is super small because I have 1047 rows per spreadsheet... rip eyesight.

For my qualitative data, I have held off observations since I cannot ethically prick myself at ASU. I guess these new, out-of-the-blue regulations are understandable, but still very annoying. I can easily take these measurements as I have set up printed sheets (with pictures) and a Google form link in my Drive to enter the observations.

My plan for the next week then is to finish collecting my data. Monday - Wednesday: perform RBS on samples (saline & canine blood, or human blood) -- I'm ready for either. And then, Thursday and Friday, finish qualitative analysis. I am confident that I will only need this week to finish data collection, and since I have been working on data analysis along the way and since I have a plan for analysis of new data, I should be set to finish in 1 week. The only question that remains is whether the liquid tested will be human blood or not.

I have no problem working in the lab and then analyzing the data at night... anything to get the best data possible!

Sorry for all the disappointing news, but we are going to pull through... with RBS tomorrow, I'm trying my hardest, and things are looking up!

Excited for next week,

Yash

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Progress: Sample Preparation, 3LCAA, and More!

Hey readers!

What a week... we are in the home-stretch of data collection! Last week, I went in the lab on Tuesday, Thursday, Friday, and all day Saturday (anything to get the data we need on time!). But, of course, my amazing readers don't care about the time I spent -- you care about what I actually did and what goals I accomplished.

So, as you may know, the goal of my project is to optimize HemaDrop™ by finding the ideal combination of coating and substrate (to create the optimal level of hydrophilicity) upon which to solidify blood drops uniformly, quickly, and cost-effectively into a film.

To do this, there are basically 3 main stages of work in the lab for me:

1. preparing samples, 
2. characterizing the surface energy of each of the samples to quantify hydrophilicity with 3 Liquid Contact Angle Analysis, and 
3. testing the uniformity of the sample quantitatively with Rutherford Backscattering Spectrometry and qualitatively with observations. 

Due to technical difficulties (the fridge becoming a freezer and freezing all of our coatings -- there was literally snow in the lab), I started this week behind schedule -- our work in preparing the coatings the week before was worthless. So, on Tuesday, I re-made the secret sauce (aka, proprietary coatings) and prepared the substrates (microscope slides and Si wafers) for application. 

Dr. Herbots said that two of the coatings looked like beer, so I posed like this... it was wild.

One big change that we decided to make was to only use 1 type of microscope slide (borosilicate -- they're so accessible that you can actually buy them on Amazon and get them in 2 days #PRIME). The reason for this reduction in number of substrates was that the soda lime slides were not pre-cleaned and not sterile. Using these slides would introduce a great deal of error and even potentially compromise our other samples. 

But, don't worry, we won't be reducing the number samples... we're actually testing another coating with a different type of metallic nanoparticle (I can't reveal the identity in this blog unfortunately, but I think I can in my research paper), so now we have 2 substrates (microscope slide and Si wafer) and 5 coating treatments (no coating, metal 1 at high concentration, metal 1 at low concentration, metal 2 at high concentration, and metal 2 at low concentration). 

So, for all of you counting (I try not to nowadays...) that's 10 different possible combinations, with 2 of each combination being made (1 for characterization with 3LCAA and 1 for blood application) -- so 20 samples! We actually made 3 of each individual slides as a validity precaution to make sure the coating was applied correctly to at least 1, so that's 60 samples, but we will only end up using 20, so don't even worry about those for now.

For 3LCAA, we went in on Saturday ready to perform some measurements, but ants had attacked our store of alpha-bromonaphthalene (a mouthful which is the hydrophobic liquid that happens to be the poison used in ant traps, so ants love it unfortunately for them and my schedule).

In this meme, the most interesting man in the world represents the most interesting hydrophobic liquid in the world!
Also, I didn't bother removing the beer from the meme, because I figured we could just call that coating ;)

Additionally, the samples are extremely hydrophilic, so when we apply the DI water drops to measure the contact angle, the water is spreading out and wetting the samples. So, we devised a plan to reverse the usual order and start with alpha-bromo and then apply the water, so the samples would not be ruined from the first liquid (water) spreading out. We'll see how that goes -- worst case-scenario, we just use each of the 3 of identical samples we have.

So, after a few hours of purging the clean room in which we perform the 3LCAA of ants, we decided instead to perform 3LCAA all day on Monday (tomorrow) and work on making sure our samples were prepared well and ready for analysis. The samples are now sitting in the laminar flow hood in this apparatus:

We mac-gyvered a sample drying rack from sterile pipettes and aluminum foil, in a minor engineering feat!

I am confident that we can finish the 3LCAA characterization by tomorrow and (LATEST) Tuesday. Then, the plan will be to, in conjunction, start applying blood on the samples Monday, Tuesday, and Wednesday (and recording qualitative observations with my updated coding sheet). Then, on Friday RBS can be performed on a majority of the samples...

However, I see data collection with RBS taking 2 or 3 sessions, so I think that my data collection could spill into the week following the February 19th deadline (probably Wednesday). I understand that this is not ideal, but since I will have all the 3LCAA and qualitative observations finished along with most of the RBS performed by the 19th, I can definitely work on data analysis in that period as we fill in any data not collected yet.

I am working as hard as I can, and I would embrace working in the lab most days and analyzing data at home in the evening. Anything for the data! Moreover, I can write my results with a few blanks for data if I have to. My meeting with Mrs. Haag is on Thursday (3/2) rather than Tuesday the week that the Results are due, so that could also potentially give me a few days to write it up.

Sorry for the extremely long and slightly serious post, but I wanted to give you guys some specifics on my progress and plan for the next 2 weeks.

Although this week was intense, it is definitely exhilarating to be in the lab working on an experiment I designed, so that's awesome. 

On a personal note, I have exercised for 7 days straight, so hopefully I can keep that up too! 

Cheers,

Yash

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P.S. Thanks so much to Grady Day and Alex Brimhall for helping me out on Saturday... I can always use the extra hands!

Working Overtime

Hey readers,

The first weekend into Trimester 3 -- it's been a wild ride. Let's start off with the Super Bowl -- yeah, I wanted the Falcons to win, and just after I snapped everyone a picture saying it's over and rejoicing at the fall of the heinous Patriot villains, the Falcons's secondary crumbled. Matty Ice turned stone cold.

At least the memes are still dank as ever...

But, hey, at least I am not a sore loser (except...just ask Kimy and Divya).

Since the Super Bowl went into overtime for the first time ever, the theme this week is OVERTIME!

After I submitted my lit review/methods combo to Mrs. Haag on Thursday, this weekend I worked on a paper with Dr. Herbots for Biointerphases journal, lit up the dance floor at Sadie's (except not at all), and finished analyzing the simulations from RUMP. The RUMP analysis actually serves two purposes, as it is part of the data for our paper and helps me plan for my experiments/data analysis in the next two weeks.

I did it though, Dwight... I did it!

Right now, I am feeling super motivated to work on and finish my data collection especially since the nature of my research (collecting data at an actual laboratory) makes it easy to fall behind and hard to catch up. Because of this my biggest struggle will be to stay ahead of schedule and communicate very effectively with Dr. Herbots. Undoubtedly, we will face some obstacles (e.g., particle accelerator down for a day, contaminated sample, etc.), but having a flexible schedule and working as much as possible to get the research done should help me face them head on.

Like Tom Brady dancing...

My primary objectives while getting my research done is to balance the interests of the lab with the time constraints of my research project and get the best help from Dr. Herbots. My weekly meetings with Mrs. Haag (Tuesday mornings are lit) should maintain the urgency to collect data in the lab and not get sucked into other experiments or projects at ASU. Moreover, working closely with Dr. Herbots will ensure my research is rigorous and up-to-par. Not trying to suck up, but I am definitely super lucky to have both of them as amazing mentors.

I am super super excited to get some concentrated work done in the lab without having to go after school, so I do not see laziness as being an issue. After all, it's more time with the HemaBaes... 

So, overall I see complications with the lab equipment/scheduling as being potential pitfalls, and staying on top of my schedule and communicating very actively with Dr. Herbots and Mrs. Haag will make sure I can maximize the time I spend in the lab, so I can start data analysis as soon as possible. Performing the simulations before the RBS, as we have, also will give us a great starting point for our experiments. Also, having the pairs of samples prepared already gives me a great head-start for our bulk measurements of surface energy and RBS. There will be a lot of repeat measurements, so once we get rolling, collecting the data will come in bursts. And, if anything happens, I'm not afraid to work OVERTIME (looking at you, Atlanta)!

Apparently, pitfall memes are a thing in Pokemon? Well, we're not falling for it!

By avoiding these pitfalls, I should be on track to finish collecting data by February 20th, with 1 week for data analysis. I can also analyze data as we get it to speed up the process (e.g., 3LCAA images of slides to measure surface energy). Now that we have the samples prepared, the goal of this week will be to perform 3LCAA to get the surface energy of the samples (independent variable). Then, once this done, the uniformity test with elemental composition can be performed with RBS.

If you're wondering what else I've been up to, I have started taking some MIT OpenCourseWare classes for math and computer science to stay sharp. Also, my tutoring game is going strong. The goal for this week is to make it to the gym every day and work on my chess game too! 

I guess this applies to this blog, not Facebook...

Things are looking up! I will keep you guys updated about my progress this week. Tomorrow, I am going to the lab for the first time since I've been released from captivity (aka BASIS). But, Tuesday, I go back to meet with Mrs. Haag (jk...that is gonna be lit).

Signing off... cheers to another great week of research!

Yash

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P.S. Miss you, miss you!