Research Internship at Osaka University

Project: Preventing Photobleaching by Intensity Modulation

of Continuous Wave Lasers

Mentors: M. Zheng, S. Kawano, K. Fujita, S. Kawata

Laboratory for Scientific Instrumentation and Engineering,
Department of Applied Physics, Osaka University, Osaka, Japan

Light microscopy is non-destructive and preferred over electron microscopy for imaging biological samples. Fluorescence light microscopy is particularly useful for tracking individual molecules or staining certain organelles, which may not be clearly visible during bright-field observation. Fluorescence signals are often limited by the effect of photobleaching, which reduces the signal intensity over time. Attempting to increase the signal by increasing the intensity of the excitation laser only adds to the photobleaching, and is not effective. Molecules undergo photobleaching from the triplet state, which has a theoretical lifetime of a few μs in fluorescence molecules such as Rhodamine 6G (R6G), used in this study. When molecules are continuously excited further from the triplet state, the risk of photobleaching increases. If the molecules can first relax to the triplet state, they can then be safely excited again to release more photons, which results in a gain in fluorescence signal without photobleaching. In this study, we modulate the intensity of a continuous wave (CW) laser with an acousto-optic modulator (AOM) at a frequency range of 0.1 – 10 MHz. Preliminary results suggest that amplitude modulation causes a decrease in fluorescence decay and increased fluorescence signal in R6G thin film samples compared to CW laser excitation. Decay rate and initial fluorescence signal were found to be correlated with excitation intensity. We find that manipulating the modulation parameters gives a frequency and wave shape that together reduce photobleaching in fluorescent molecules. Incorporating this modulation scheme into fluorescence microscopy should significantly improve fluorescence imaging for biological and other applications.

PICTURES: LaSIE Welcome, Farewell, and Everything In Between

Hello OSAKA!!! Taking the shinkansen from Tokyo to Osaka to begin my internship at Osaka University	First night welcome with lab mates and fellow NanoJapan participant, featuring Osaka-style okonomiyaki, soon to become my favorite Japanese dish.	First lab picture after my welcome dinner – the beginning of my philosophy to try every food that was put in front of me, including LOTS of SUSHI!!
My experimental room for the summer – laser, photon counter, mirrors, filters, computer, and more. I worked closely with Meiling-san, a Chinese graduate student.	Prof. Kono from Rice University, NanoJapan, visits my lab and I show him around my experimental room.	My Australian professor and his Japanese wife welcome a Chinese exchange student and I into his home to learn how to cook some Japanese food.
It is a tradition in Japanese culture to bring back gifts to friends and coworkers after returning from a trip. Sweet red bean stuffed treats from a weekend trip to Hiroshima, Japan.	The excitement that these sweet treats bring to the lab!! These are two of the very few girls in the lab.	Spreading Georgia Tech paraphernalia all the way to Japan! My end of the summer gifts to each lab member.
I decided to dress up in traditional Japanese attire for my farewell dinner, a yukata (robe) and obi (sash). The obi took about 10 minutes for a Japanese girl to tie for me, with references to instructions.	The whole lab showing up to my farewell dinner. To my right is my assistant professor and immediate mentor, Fujita-sensei.	Final lab group picture after my farewell dinner. Sitting to my left is the head professor in the lab, Kawata-sensei.
Participating in a tradition at Japanese barbeques – smashing a watermelon open with a stick after spinning around in circles.	Japanese guys trying Jelly Bellies! The jalapeno flavor was the most intriguing and amusing to watch them eat.	Last day in Osaka with my friends from lab…

Week 4: First week in lab…

I met several of the students on the Sunday that I arrived, and they showed Catherine and me around the area by our apartments and took us out to dinner. On Monday, we were taken to lunch by Catherine’s lab, and then I went to my lab for the afternoon. Two of the graduate students helped me to make a computer account, name card, photo, and showed me around several of the experimental lab rooms. I did some background reading out of a textbook, and was taken to dinner for my “welcome party.” This was quite an experience, and I think probably the best welcome I will ever have to a new workplace. There was so much food being ordered, and everyone wanted to make me try everything and hear my stories. I had a lot of fun and got a chance to talk to several of the students somewhat in depth. Everyone has been very hospitable and seems to be enjoying having me in the lab.

As for my research project, I basically jumped right in and I’m learning as I go. The goals are fairly clear. I had my first meeting with my professor and his graduate students on the second day in lab. They gave me some background information and we discussed my project and the major steps and goals. I will give a presentation in two weeks at a smaller group meeting regarding my progress so far.

I have definitely observed the hierarchy present in Japanese labs. Under Professor Kawata, I have one associate professor that is overseeing my project, but a few of his upper graduate students are actually helping me to design everything. Finally, I am working as a peer with one of the newer students to design the microscope set-up for my experiment. All of my mentors at each level have all told me personally to come to them and ask them questions at any time, so I feel like I have plenty of resources. A few of the Australians who obviously speak very good English have helped me with issues other than research problems. I have enjoyed talking to many of the students, although it’s sometimes a struggle for the youngest students to carry on an extensive conversation in English.

I was highly impressed with the amount of English spoken in the lab. There are several other students/professors who are international and do not speak Japanese as their native language. All lab meetings and presentations are conducted in English. If I have a question, I can definitely ask almost any of the upper students or professors and be easily understood and get a comprehendible answer back in English. I will have to make an effort to actually use my Japanese since it is rather easy to speak in English and be understood. I don’t think the language will be much of a barrier, if at all, to me working effectively (except for the computer programs and menus written in Japanese).

My housing is great, and I have lots of space (probably more than I need). It is definitely very convenient to have my own kitchen and bathroom. I doubt that I will meet anyone living in the same apartment complex, but that’s ok. It is a good location, close to a monorail station and to my lab, although it’s quite a walk to the most useful subway station. This is probably the most space I’ve ever had purely to myself, since I usually share an apartment with other girls at my university in the US. The commute is a 10-15 minute walk to my lab from my housing.

My goal after the first week is to continue to build my understanding of all the concepts involved in my research. I already have an extensive collection of Wikipedia articles about every new word that I have heard. I also want to travel as much as possible, and have discovered that many of the students in the lab like to talk about their hometowns – Nara, Hiroshima, etc. – so I am hoping to have them tell me where to visit in those places or perhaps take me there themselves.

I am loving Osaka – I got the chance to explore the Osaka castle area this weekend. My lab has been great as well. I am enjoying the mix of new concepts and familiar skills that I can take advantage of already knowing.

Week 5:  Research Project
Research Project Overview:

For biological samples, light microscopy is preferred over electron microscopy. However, the resolution and signal in light microscopy are limited by factors such as photobleaching. This is a major problem in fluorescence spectroscopy. To improve the signal-to-noise ratio, the simple solution would be to increase the intensity of the excitation laser. At higher intensities, however, molecules are excited more easily to higher energy states. Molecules at higher energy states are more likely to interact with other molecules such as oxygen and become photobleached. Much research has been done to attempt to prevent this interaction to decrease photobleaching. Photobleaching can occur from many different states, depending on the lifetime of the molecule, but mostly occurs from the triplet state, where the molecule stays for a few microseconds. This project proposes modulating a continuous wave laser to create a pulse-like laser with a function generator and acousto-optic modulator. This will allow us to not shine the laser when the molecules are in the triplet state. By letting them relax between excitations, we can avoid overexciting the molecules and causing photobleaching. We can vary the intensity with an ND filter and move to different locations on the sample with a moving stage. It has been previously reported that a pulsed laser instead of a continuous laser could prevent some photobleaching, but we hope to achieve a similar effect still using a continuous wave laser. By changing the pulsing based on the lifetime of the molecules, we can measure the decay of fluorescence over time and observe less decay indicating less photobleaching. My project will be divided into three major steps. First, the fluorescent samples must be prepared using a fluorescent molecule like rhodamine 6G, a solvent and polymer matrix. The absorption and fluorescence will be measured to determine the optical properties of the sample. The second step will be to modulate the laser to create the desired pulsing effect. Finally, I will measure the decay of fluorescence to see a difference in photobleaching. If time permits, I will also try to experiment with other fluorescent dyes used in actual biological staining. If I can easily find the parameter of modulation to best prevent photobleaching, I may have time to incorporate this into a functioning microscope and also learn how to stain and image cells.

Research Goals/Objectives:
1. Learn how to operate and set-up equipment for optical measurements

2. Learn how to analyze results to determine effect of lasers on photobleaching.

3. Investigate the optical properties of fluorescent molecules.

4. Model the effect of wait time between excitations on photobleaching.

5. Test and validate model with experiments on additional molecules or with varied parameters.

Week 6:  Research Labs in Japan vs. US

As I stepped foot in the Kawata lab, I knew this was a special place. Not only am I in Japan, where the people are so helpful and kind to newcomers, but I’m also in a fantastic place to do research. Here they created the nanoscale bull structure and the first optical laser pacemaker. There’s a wonderful blend of physics, biology, and chemistry, so that any researcher can add their knowledge to the mix and learn skills from several other disciplines. The lab has the expected tight knit Japanese culture, but also an awareness that they are competing in an international research arena that requires a greater sense of purpose (and English language skills).

In Japan, people-watching can reveal so much about your surrounding environment. The lab members have unique mannerisms and methods for communicating and working that are inherently Japanese. When addressing each other, older students usually call the younger students by “kun” and professors, of course, by “sensei.” Nicknames usually come into the mix, making learning actual names a little confusing. I think that perhaps the foreigners still remain slightly outside the circle, at least for a little while. For example, I find that I probably retain more respect than I perhaps deserve since I am still the new student from America.

The international focus in this lab creates a whole new dynamic that creates a great divide between informal casual conversation and professional academic conversations. The language for socializing and casual conversation in the lab is Japanese, often leaving me left out from these conversations (although it’s easier to get work done when I can just tune out all the Japanese conversations). When the students are forced to give presentations in English during lab meetings, their confidence level drops significantly, especially when asked questions in English. Their English is impressive though, and I can’t imagine how tough it is for the students to do this. I think the students should probably take better advantage of having several native English speakers in the lab to practice answering questions and improve their presentations by asking ahead of time, but they are often too shy to do this. For example, we have paper review sessions every week, with three new journal papers presented by the younger students to the entire lab. I was surprised to see that one of the papers was from the lab that I’ve been working in at Georgia Tech. Someone told me that they told the student presenting this to come ask me about the paper, but that never happened. I was intrigued by article posted on the NJ site about the use of English in Japanese research labs. The conflict between the focus on English hindering scientific progress and the need for English to communicate well in the scientific world is a difficult one, but I think it easily mirrors other such conflicts on encroaching Western culture on Japan. I think I am a little biased to address this situation, but I think the use of English will enhance the experience of the students in the lab, allowing them to have more opportunities in the world in science or otherwise and letting the lab be more recognized with more impressive publications in English.

Communication styles in Japan take a little adjustment for enthusiastic verbal Americans. In any situation in America, I am often by far the quietest and least outspoken, but here, the Japanese certainly have an even quieter demeanor. I am not much of a joker, at all really, yet one of the students commented that I apparently find everything funny? I guess it’s just my attempt to be lighthearted and ask questions to understand everything. They’re not huge talkers, and actions are often more credible than words. Their explanations are often extremely indirect and difficult to understand, especially when their English grammar isn’t perfect either. It recently took about three people telling me about the same situation before I finally understood what point they were trying to get across. I had one student tell me that a professor wanted to meet with me because he was going away, so I figured this was about research. Another student tells me that he and the professor want to plan a trip somewhere. Finally, after more discussion, I determined that they wanted to find out which date would be best for me and where I would like to go if they planned a weekend day trip. After several roundabout conversations, we finally were all on the same page. I found that some of the Japanese do not have good eye contact when talking. I found this particularly difficult when I was in a big room of talking people and was trying hard to distinguish who was talking to me, and whether they were talking in Japanese or English. Without eye contact, I was confused and it took much longer to get my attention and concentration.

The idea of saving face and not confronting older members of the lab is a little difficult to deal with coming from the American culture. With the addition of being uncomfortable speaking English, this becomes even more evident. In paper presentations, it is clear that only the professors and most distinguished lab members will ask questions from the students. If the student can’t seem to understand the question because it is asked in English, or can’t come up with an explanation in English, they are basically excused from the question. In lab meetings, presentations are given smoothly in English, but there is little discussion among members, and never questions from younger students. When I am working, to me the most obvious way to learn is to always ask questions and ask for explanations, but this isn’t exactly the norm. This bothers me a little bit, because I have found myself wanting to speak up and ask questions in meetings. I either feel bad because I know it is already difficult for them to answer in English, or it seems completely out of place to ask questions of those older than me, since I am clearly one of the youngest and probably least experienced in this field in the lab.

Other common ideals of the Japanese workplace are evident in this lab. Thorough documentation is required, so that the content speaks for itself. Biweekly reports are written and submitted for the group to read and comment on. There is definitely an atmosphere of patience and slow and dedicated work. I am never expected to understand something right away. It seems impossible to them, like they expect a slow learning curve to be a requirement. This can be a bit frustrating, when I am told just to read the manual again for equipment that I think I know how to use and implement in the system. Patience, thoroughness, and conformity seem more important than speed or creativity, which is quite different from the situations I am used to. For example, in a lab in America, all presentations given at lab meetings had a clever and witty title and often used interesting analogies to grab the attention of the lab members. Here, I could never see that happening. The atmosphere is much quieter with a straightforward following of the rules.

Overall, the Japanese research environment emphasizes hard work, long hours, and a sense of team. When I discussed my upcoming presentation with a professor, I commented that both I and my grad student advisor were assigned to give presentations, but we had basically been working on the same exact tasks. He just laughed, and said that the American student in the lab last year had the same concern, but the Japanese don’t think anything of it. We are raised in an academic environment that encourages individualistic ideals and a competitive need to show off exactly what we’ve done as individuals. In Japan, the sense of team is much greater, and they aren’t concerned about this. In American labs, compared to Japanese, I think there is more focus on progress and performance than dedicated time. In Japan, it seems that sometimes time, loyalty, and age are more respected than particular ability or knowledge. For example, younger students would never be expected to know more than an elder. In contrast, in my lab in America, we have a diverse group of computer engineers, electrical engineers, computer scientists, and biomedical engineers. Often the young biomedical engineering students are asked by older students or the professor about bio-related facts or suggestions regarding their knowledge from biology classes. While the lab in Japan obviously emphasizes written reports and oral presentations, the background and training in these subjects is quite different. Japanese students have never given a presentation until being part of a lab, where American students practice presentations from middle school, high school, and college.

Which lab structure do I prefer? In Japan, sometimes the slower pace is difficult. I always want to move faster so I can learn more in this short amount of time. I think that sometimes the strict structure of respect can limit the progress of science. I do like the sense of team though. The lab is a community. People like to interact with others’ projects. I wish the younger students would speak up to ask questions, although I’m not sure if this is due to English language or just the culture. It is nice to have the greater expertise of having so many associate professors in the lab, and not relying only on the main professor for the ultimate research questions and decisions.

Week 7: Preparation for Mid-Program Meeting in Kyoto

It is hard to pinpoint my biggest personal accomplishment at this point. Nothing really stands out in my mind while I’m still in the midst of all of it. I am probably most proud of what I’ve learned about optics in the lab. While this may be more of a research accomplishment than a personal one, it is rewarding to feel like you understand what’s going on, be able to follow a discussion on your topic, and contribute insightful conclusions as well. I gave a successful presentation after only two weeks in lab about my project, and with that going well, I was happy with what I had accomplished in even such a short time. I am in no way satisfied with what I know of course, and I still try to learn something new every day. I also enjoyed showing my parents around Japan without getting too lost. It was a change to be the most knowledgeable about something and I was forced to use my Japanese without anyone to help me.

My biggest personal challenge has been trying to push myself to learn more about the Japanese language. My language skills were probably at their height at the end of the three week orientation classes. My classes here in Osaka didn’t start until July 1^st, and even then they started basically at the beginning, so it’s mostly review. The class is worth it though, I think, but sometimes a little boring. I can pick up on vocabulary that I missed the first time around that now seems easy. I have a hard time remembering to use Japanese with the students in lab because they always speak to me in English and will understand me if I speak English. I will try to continue using more Japanese in lab, and I do use it a lot when out sightseeing. In terms of language proficiency, I think I have the hardest time with understanding what people say in Japanese, which makes it difficult to have a conversation or learn from listening.

My research is progressing forward quite well. I think I am maybe a bit behind schedule, but even so, things are picking up. Our optical system is complete, with every little piece of equipment functioning correctly. We have successfully measured progressively more difficult samples, and are working towards being able to get the data I need for my experiment. Every day raises more questions, and slowly, more answers. There is definitely not enough time left, but I will still have accomplished much when I finish. Having to write research reports every two weeks keeps me on track of analyzing what I’ve done so far, so it should be easy to compile my final report from these.

Week 8: Overview of Kyoto Weekend and Research Update

I really enjoyed the chance to be together with the group again. Even after a month apart, we picked right back up on our friendships and discussions. It was nice to have convenient travel/sightseeing partners again without trying to ask Japanese students to go with us.

As for the Mid-Program Meeting, I thought the discussion was helpful to hear some of the other students’ experiences, but I had already heard a lot of the comments from the students directly. The people that I hadn’t already heard from didn’t really speak up. Although people might not have enjoyed being forced to talk, I feel like at least one question should have been addressed to each person so we got to hear a little something from everyone and certain people didn’t dominate the discussion. It made me appreciate some of the great things about my lab and realize that other students had some of the same difficulties as well.

The IORI workshop was a really great opportunity to be introduced to all these arts that we couldn’t really do any other way. I particularly enjoyed the calligraphy – so cool to take home the work that we did during practice and on the final fans. The kyogen performance and workshop was probably the highlight of the day, and I would highly recommend keeping that as part of the experience. Of course, I highly enjoyed the tea ceremony, Noh drama, and martial arts portion as well.

The Machiya stay was perfect for our group. Having the two houses let us see two slightly different styles, including the bigger house, and the one with the view of the river. It was a nice setting to allow us to hang out as a group at night, rather than being in separate rooms in a hotel. It also gave the weekend a feel of being in traditional Kyoto.

The only challenging things about this weekend were sitting on our knees in the tea ceremony, being patient enough to wait for geishas to come out of tea houses at night in Gion, and trying to fit all the amazing things to see into our limited time!! I am lucky to be so close to Kyoto in Osaka, and also to have some Japanese friends who want to show me around the city more in the next few weekends. The highlight of the weekend was probably going to the Fushimi Inari shrine on Saturday night. It was such a different experience than visiting the other temples – going at night and having the place basically to ourselves. It had a bit of a spooky feel but also very beautiful lit up at night. Other highlights were the IORI arts workshop and finding an amazing 3000 yen deal on yukata + obi + shoes + bag! All the girls and even the guys went home with matching bags from this yukata shop.

Prof. Kono’s visit to my research lab gave me a chance to try to explain my research to someone other than my immediate lab members who are familiar with the work and processes. I also enjoyed joining him on the lab tour and learned some new things about the work going on here that I had not previously heard. I am probably behind on my projected research timeline, but I don’t think the next phase will actually take as long as I had given it on the timeline, so I think it will be ok. Everything is finally working, which was the major bottleneck of time. From here, I just need to put in the time to get all the measurements done and hopefully see the desired result. I have had several lab members critique my abstract, although I may have to cut out some of the projected results since I don’t see myself having those down concrete by next week when the final abstract is due. The poster should be fairly easy to put together, although I think I should figure out a few more things to get pictures of for the poster. For an actual research update, I have attached my report to the lab written last Wednesday right before Prof. Kono came in addition to some powerpoint slides that I showed him. I have since resolved a few of the problems discussed in that report, so I think things are going well.

Time is flying by and there are only a few more weekends to squeeze in everything to do! My lab has planned a trip to the Ise Shrine, and I plan to visit Kyoto again for one or two days. Hopefully I will have the chance to attend the Gion festival in Kyoto on Thursday and also a festival in Osaka the next weekend. This weekend I plan to either go to a baseball game, Bunraku performance, fireworks in Kobe, or maybe Universal Studios in Osaka – all of these if possible!!

Week 9: Reflections on Japanese Language & the Role of Traditional Culture

My language experiences have made me feel frustrated, awkward, and confused, but not necessarily challenged. I’ve always found a way to do what I want to do without being hindered by language. I guess I’ve gotten used to getting along without speaking so much of the language. Not that this is a great motivator, but it’s practical… It doesn’t intimidate me anymore like it did coming into Japan the first week or so. I still highly value learning as much Japanese as I have, and it’s certainly been useful.

At this point, it has become fairly routine to deal with ordering food, buying things in shops, and asking for directions in Japanese. These basics get me through my everyday life. In my research lab, all science-based discussion is conducted in English, especially for me at least.

Listening is probably the most challenging area of dealing with Japanese. I feel pretty helpless when people in restaurants or shops start talking to me in Japanese without any attempt to communicate in English. There’s not much I can do except shrug and try to say in my little Japanese that I don’t understand. Body language and gestures can say surprisingly a lot when necessary. When I was taking my parents to restaurants, it was one of the first times (other than being alone but I don’t really eat out alone) that I was without the help of any other students or Japanese friends. There was more pressure than usual to pull out any Japanese that I could to make things go smoothly. I managed though, and we got our tasty food successfully. The most awkward situation probably comes from sitting in the common room in lab and listening to all the Japanese guys chatting and joking, and having basically no idea what they’re talking about. Although, if I’m really interested in understanding the conversation, one of them will usually stop to explain what’s going on to me in English.

Reading Japanese manuals and computer error messages and menus in Japanese can slow down research a little bit, but I’ve just gotten good at searching the internet for similar or equivalent manuals in English, and comparing my English versions of the program to the Japanese version to understand it better. Menus in restaurants are another one of my roadblocks, especially when it’s all kanji and no pictures. At lunchtime, I usually rely on my helpful and generous Japanese lab mates to translate for me and make suggestions.

To continue studying, I have tried to learn a little kanji to help reading signs. I also try to learn more specific phrases that I can remember to use on a daily basis, rather than just long vocabulary lists. My survival Japanese class at university has been useful to review and catch simple things that went by too fast when studying in Tokyo. In the lab and around on the weekends, it’s more like continue practicing rather than studying.

As for future language study, I’ve considered for a while taking Japanese classes at my home university, and I am now much more likely to do this. If I don’t take actual classes, I’d like to at least keep up studying as a hobby. I hope to return to Japan at some point, whether for a long term or just a short term visit, I’m not sure.

It’s amazing how fast things can turn around. We had two days of terrible measurements, and I was about to give up and spend time discussing the problems. After restarting equipment a few times, waiting patiently, and by some other unknown miracles, it’s all working again. So maybe there is hope that I can get some concrete results done in the next few days.

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