Stories & Features

Interview with Dr. Hari Shroff

Dec 19, 2015 by IDEX Health & Science

IDEX Health & Science: Beginning undergraduate study at the age of 14 to already becoming a Tenure-Track chief at NIBIB (National Institute of Biomedical Imaging and Bioengineering)’s section of high resolution optical imaging laboratory—we would like to hear about your journey so far.

Dr. Hari Shroff: Sure! I entered the University of Washington at an early age through this special program that they still have today, actually—where they take fifteen or sixteen kids every year, and you do kind of intensive study in a “transition school.” If you complete that, then you’re accepted as a full-time student at the University of Washington. So that’s how I got in early. Now I think a huge part of my continuing in scientific research was that I was lucky enough to work as an undergraduate researcher in a lab in the Chemistry Department at the University of Washington. That was really transformational because I was able to see that real research isn’t done just by taking classes. It’s kind of messy, and one has to get into the lab, and the answers are not always straightforward. I realized that I really enjoyed the feeling of knowing that, if I wasn’t invested in this research into the unknown, then maybe this wouldn’t get done. We were trying to do things that weren’t written in the textbooks. That’s how I got the “research bug,” I guess you could say.

After that things kind of progressed: I went to graduate school at UC Berkeley in biophysics—because I couldn’t really make up my mind about what I wanted to do after my undergrad. I liked the interdisciplinary research that I was doing as an undergrad which was partially chemistry, partially physics, partially biology. I figured if I did a graduate degree in biophysics I could continue that. So that’s when I got into single molecule microscopy. I worked for a guy—Jan Liphardt— who had been studying single molecules of RNA. He had just finished his postdoc and had just started his lab. I was his first graduate student. He’d just gotten his position at Berkeley, and initially my goal was to do some real science. But I found that, despite my intentions, I ended up in tool development and in tool building. I worked on constructing a microscope that let us both pull on molecules of DNA and observe the single molecule fluorescence.  So that was kind of my entry into microscopy.  Actually I built a TIRF microscope before you could buy them.

IDEX Health & Science: Oh really?

Dr. Hari Shroff: Yes, and this would have been the early 2000s; they were just starting to come on the market. In order to isolate the fluorescent emissions from a single molecule without background—TIRF was the sensible choice. So I built a magnetic microscope with tweezers in place of the bright field pillar of your microscope. Then below the stage there was a high numerical aperture objective so we could do objective side TIRF. Actually it was the first time I realized the value of getting good optical filters, although that predated Semrock, I think.

IDEX Health & Science: Semrock was founded in September 2000.

Dr. Hari Shroff: I was, at the time, trying to find the best filters I could from Omega Optical, which used filter technology that has now been replaced by your technology.  That’s when I realized the value of very good filters for isolating single molecules against a huge background. So the TIRF was part of it, and the filters were the other part. I built this microscope, and then I designed and calibrated these probes. The idea was to build single-molecule force sensors. It’s funny because that idea was a little ahead of its time. I wanted to put these things inside cells, but I didn’t understand anything about cell biology. I only got as far as making these sensors and putting them in vitro DNA loops to measure stresses and strains. That original paper has been cited a bunch because now force-sensing is a hot topic, along with protein polymer sensors and with FRET dyes.

But I left that behind because, as I was finishing my PhD and starting to think about what to do next, Eric Betzig rolled through Berkeley. He was actually talking not about PALM and STORM, although he had hinted that he had this great super-resolution idea, he was instead talking about this idea he had for doing lattice microscopy. I was very taken with his talk.

I have always been very fascinated by the fundamental mismatch in size between what a biologist wants to see and what they actually can see. He gave this talk and he was talking a little bit about super-resolution, and I wanted to drop what I was doing and immediately work for him. It was about six months after Betzig’s 2006 Science paper came out when I was lucky enough to join his lab—I was one of his first two postdocs. And that’s how I really got into hardcore microscopy development.

I was one of the first people at Janelia Farm, and I got there shortly after his PALM paper came out. So instead of working on this thing I was originally going to do, which was this lattice microscope, I got into PALM. It wasn’t planned, but it was very good for my career. Super-resolution was kicking off in a big way with his paper along with a lot of other work people were doing, and I had the chance to do a lot of the early developmental work. Once again, I built a TIRF microscope. Or, rather, I think at this time Olympus had a TIRF illuminator, so I used that. But I did a lot of work optimizing the sample prep and doing multiple color PALM and live-cell PALM—all in two dimensions. That’s when I learned a lot about the “nuts and bolts” of microscopy: what makes a good microscope, design choices , optical design, this and that which I mostly got from Eric Betzig. As a graduate student, I was just mostly on my own playing around. So I made a lot of mistakes in the TIRF microscope I built in grad school. I studied Dan Axelrod’s papers and tried to copy them, but it wasn’t until I met Eric that I really learned how to do it right.

Then I was at Janelia Farm for two and a half years, and both Eric and I were getting tired of PALM. And then it was time to move on anyway to start my own lab. Then an opportunity at the NIH (National Institute of Health) came up. In working for Eric I realized that microscopy was a very rich field, that cell biologists really needed microscopy, and that brought me to the NIH. Instead of working on PALM and STORM—localization microscopy—I made the conscious decision to work on microscopes better suited to the study of live, dynamic samples. At least, over the last five years, my work has been much more centered on microscopes used for live imaging, including structural illumination microscopes and also light sheet microscopes. So that’s how I got to where I am!

IDEX Health & Science: You briefly touched on how super-resolution microscopy has taken off. Overall, how do you see the growth in this field?

Dr. Hari Shroff: Most of the “low-hanging fruit” has been picked, and all the proof of concept work has already been done. Biologists are convinced that these techniques can be useful for their research. But I would say that there is still a long road before these techniques become as easy to implement as widefield microscopy or as confocal microscopy. There’s always going to be this fundamental hit in signal-to-noise or in speed, because your pixels are much, much smaller in super-resolution microscopes. I would say that the dyes haven’t quite kept up with the capabilities of the microscopes, so the whole field is driven by dye development. The Nobel Prize was given for developments in chemistry where people used these switchable dyes for their on and off states. The reason it was given in chemistry is because these super-resolution techniques that break, say, 100 nanometers rely on the properties of the dye more than the instrument. Fundamentally, the reason why we can’t go as fast as we can in conventional imaging is because you’re putting  a much greater burden on the dye. You’re asking for a lot more information from the dye molecules before they bleach. That’s challenging. I would say that I’m very optimistic about super-resolution microscopy, and these techniques are starting to become part of the tool kit—right—but there’s still room for a ton of development.

IDEX Health & Science: Here is a picture of a “microscope” that you are currently developing. This does not look like any conventional microscope used for biological imaging. Would you like to talk more about this?

Dr. Hari Shroff: Although it doesn’t look conventional, I would say that that kind of microscope is quickly becoming conventional. The microscope you took a photograph of is an example of a light-sheet microscope. There are actually a couple of reasons why it looks funny: one is that it’s opened up—it’s not black box in a way that you would look at a commercial microscope. It’s all open for the user to see where all the optical components are and how they’re arranged because we’re at a technology development lab. The other difference is that instead of having just one objective, that microscope has two objectives. The objectives are set at 90 deg for doing light-sheet microscopy. So that is probably the more peculiar thing about it. I think you can see these two objectives that are over the sample. The sample is kind of mounted conventionally in an inverted microscope base, but then we use these two objectives from above to introduce light-sheet illumination and to collect the fluorescence generated from the light-sheet. That’s why it looks so strange.

IDEX Health & Science: And this is mainly to improve signal-to-noise ratio in the sample?

Dr. Hari Shroff: Yeah—effectively they make better use of the light budget. The key idea is that if you can match the illumination volume to the detection volume—which most microscopes don’t—you can make much better use of the fluorescence you produce and on the light that you shine on the sample. All living samples have limits as to how much light they can take, and it’s kind of shocking when you realize that in conventional microscopy you typically subject the sample to the light of multiple suns. Light-sheet microscopes much better approximate natural conditions because a much greater fraction of the fluorescence that is generated is detected, although you’re still only using one objective to detect, typically, so most of the fluorescence is still wasted. Most microscopes are amazingly inefficient when you consider the small fraction of the solid angle that they detect.  However, light-sheet microscopes are better than most because now your illumination is relatively well-matched to your detection, whereas in most other microscopes you illuminate much more of the sample than you detect.

IDEX Health & Science: How do you see the role of thin-film optical filters in advancing your research work?

Dr. Hari Shroff: A game changer. The reason that people can build great microscopes and take full advantage of developments in fluorescent protein technology or synthetic dye technology is because you can cleanly separate—in a fluorescence microscope—the excitation from the detected light. Also the fact that filters like the ones you guys make are so robust means that they can be used essentially forever. It’s very difficult to damage them. You can achieve a very high signal-to-background ratio, actually matching the capabilities of your microscope and dye, to collect such great images because of this crucial component of the fluorescence imaging train. So I would say that new developments are transformational in fluorescence microscopy.

IDEX Health & Science: Apart from conventional academic publications, you are also actively involved in patenting your designs and methods. What is your view on patents, and do they foster academic-industry collaborations?

Dr. Hari Shroff: There are always going to be some people that are unwilling to build their own microscopes. That segment of the market might be changing or there might be more people who are willing to take risks and build their own microscope. But there’s always going to be some people who want “turn-key” instruments. As a developer, if you have a good idea, you try to publish the proof of concept, do some follow-up biology work, and make your tools as transparent as you can. But if you want to get a company to commercialize the product, which is how it’s ultimately going to reach the most number of biologists without the technical training to build their own system, often companies are interested in protecting their investment in your technology. Patents are one way to accomplish this.  I take as multidisciplinary an approach as I can, so I disseminate my work as completely as I can so that builders can copy what I do and improve it – although I’m sure I can do even better. But I’m also interested in working with companies to help them adopt what we do, so that people can just buy instruments  and they can worry less about the alignment or the repeatability. You don’t have to be a trained optical engineer or physicist in order to operate it. Intellectual property has its place in that sense.

IDEX Health & Science: You mentioned working with Eric Betzig, and he won the Nobel Prize in Chemistry during 2014. What was your reaction?

Dr. Hari Shroff: Well I would say that that the names are about right. Those guys definitely contributed in a seminal way to the field. I was surprised that it happened when it did. I would have expected giving them the Nobel Prize for super-resolution in about another decade. The reason I say that is that there have definitely been some applications, some specific examples of things we’ve learned because of these microscopies, but those are relatively few and far between because the technologies are still young and still developing. I don’t have any particular insight into, of course, how they award Nobel Prizes. I figured maybe the Nobel Committee would have waited until the field matured a little bit, but the names are about right.

IDEX Health & Science: Do you feel that there is more expectation of you coming from Eric Betzig’s lab?

Dr. Hari Shroff: Because I worked for him?

IDEX Health & Science: Yes.

Dr. Hari Shroff: I don’t think so. I was personally involved in the growth of super-resolution, but only because I was lucky enough to be at the right place at the right time. I did a lot of developmental work, but the seminal stuff all came before me. I don’t feel any particular pressure in that sense. I do think that it’s a good time for microscopy in general. Super-resolution microscopy is certainly enabling and getting a lot of press, but there are other microscopy techniques that are leading to discoveries and allowing us to image specimens that would have been unthinkable ten years ago. In that sense I’m excited because I feel like this prize elevates microscopy everywhere.

IDEX Health & Science: During the last several years you have been Co-Director of Optical Microscopy Course at the Marine Biology Lab at Woods Holes.  How did that come about?

Dr. Hari Shroff: My introduction to the Marine Biology Lab was actually as a student in the physiology course—which I would recommend to anyone who is interested in cell biology because it really had huge influence on me. In between graduate school and my postdoc with Betzig, I realized that microscopy was fundamental to cell biology. I took this course there—a very intensive summer course—where I was working hand-in-hand with a lot of the experts in cell biology. The following year I went back with Eric Betzig—we took our microscope actually, the PALM system that I built with him at Janelia Farm. Ever since then I’ve been involved in the Marine Biology Lab in one way or another; you kind of develop a fondness for it after you’ve suffered through a summer as first, a student and then as a TA. Those were very heady, exciting times, but also sleepless times. Something very special happens there during the summer when you have these world-class scientists congregating for a couple of months. You end up with these collisions which are just difficult to have otherwise.

People have this kind of “can do” attitude about science, and it’s also a great place for microscopy because some of the world’s best microscopists usually hang out there during the summers. Anyway, I’d been sort of going after that TA-ship and gave a few lectures here and there in different courses.  I was invited to give a lecture by Bob Hard (the other director of the course), and then he kind of sprung up on me that they were looking for another director, and would I consider doing it. My first reaction was: This is going to take me away from my lab! This is going to be a ton of work.  One of the privileges of being at the NIH is that I don’t have to teach classes, and so I figured that this would be a way of giving something back to the community. It’s also fun and a good break. I actually get some of my best ideas just from daydreaming and talking to students. There was kind of a selfish motive there, too. But I think it has been useful, and it has been impactful on these students who might not have much prior microscopy experience. They come and get a crash course from us. I don’t know how long I’ll keep doing it—because it is a lot of work—and I think it’s good to have turnover and “fresh blood” in these courses as well. But it’s definitely been fun, and I’ll be there again this year.

IDEX Health & Science: Thank you, Hari! We’ll look forward to seeing you at the MBL Course later this year.