Interview with Bruno Pichler

Bruno Pichler studied medicine, obtained a PhD in neuroscience, worked in the labs of Arthur Konnerth, Tom Mrsic-Flogel and Troy Margrie, and was R&D manager at Scientifica, before founding his own company, INSS, “to provide the international neuroscience community with bespoke hard- and software solutions and other consulting services”. He is not only a highly experienced builder and designer of two-photon microscopes but also a very friendly and open human being. So I was very happy to have the opportunity to ask him a couple of questions.

The interview took place on September 8th 2020 in a virtual meeting and lasted around 1.5 hours. Afterwards, I transcribed, shortened and edited the recorded interview. For a brief orientation, here’s an ordered but non-exhaustive list of the topics discussed:

Why neuroscience?
How to get into optics and programming
Role models
Projects in academia
Why didn’t you become a PI?
At Scientifica
Founding one’s own company
Performance checks for a 2P scope
How to clean optics
The detection path
Teaching people how to solve problems
Fixed-wavelength lasers
Multiplexed acquisition
Advice to young scientists
Bluegrass music

If you find any questionable statements, you should consider blaming my editing first.

And since the entire interview is quite long, take a cup of tea, and take your time to read it


Peter Rupprecht: You studied medicine before you started your PhD. What was your motivation to do a PhD in neuroscience, and to continue in neuroscience afterwards?

Bruno Pichler: This was just something that happened: I really loved the first years of medical school, basic science, physics, biology, anatomy, physiology, and all that. But halfway through medical school when the clinical work started, I realized that it wasn’t for me. So I looked for new inspiration, and I stumbled upon the website of Arthur Konnerth’s lab. I called on a whim and half an hour later I was in his office, and he offered me a job. He said, “why don’t you take a year off from medical school and start working towards a PhD here?” So I worked full-time in the lab for a year, and then went back to finish medical school. But at that point I had no interest in a career in clinical medicine, I just wanted to complete the medical degree and then work in the lab and continue my PhD. – So, not much thought behind it, it’s just how things transpired.

(c) Bruno Pichler

PR: The medical studies did not really prepare you well for the more technical aspects of what you have been doing afterwards. How did you learn all of this, for example optics, or programming?

BP: Again, it was just something that happened: there were microscopes that needed troubleshooting, there were things I didn’t understand, and every time I didn’t understand something, I tried to find more information about it. Some of the information sticks with you – and that’s how you learn and how you get into optics and software.
For example, there was some custom-written image analysis software in the lab and I didn’t really understand what it did to the data, so I sat down with the guy who wrote it and asked about all the calculations it made and then I cautiously started to make some changes to it – and it just naturally emerged from there. I never consciously sat down and said, “oh, I want to learn programming!” I had a problem in front of me that I needed to solve, and so I solved it. And whatever I learned while solving it is now part of my knowledge.

“People who inspired me were often those in support roles, like the lab technicians who taught me how to pipette, or the engineers in the electronic and mechanical workshops.”

PR: So would you describe yourself as a problem-solver?

BP: I think that is probably an accurate description. My main driving force is that whenever I see a technical problem, I want to find an elegant solution for it. I can’t help it.

PR: Did you have a role model as a scientist, or somebody who inspired you to continue with neuroscience?

BP: I definitely have a few people who inspired me, but I wouldn’t say I had a ‘role model’. There’s obviously the intellectual giants like Richard Feynman or Horace Barlow. Then there are the scientists that I worked with, Arthur Konnerth, Tom Mrsic-Flogel, Troy Margrie, and of course all the colleagues in those labs. But, on a very practical level, people who inspired me were often those in support roles, like the lab technicians who taught me how to pipette, or the engineers in the electronic and mechanical workshops. For example, Werner Zeitz, the electronics guy in Arthur Konnerth’s lab, who is known for his famous Zeitz puller ( We were building a two-photon resonant scanner with Werner back in 2004/2005, and he built a 19” rack-mountable device box – no labels on it, just unlabeled pots and BNC connectors – which transformed the scanning data into a TV image and sent it to a frame grabber card. Nowadays, we do this in software but it was all done in hardware at the time. Same with the mechanical guy, Dietmar Beyer: He was such a skilled manual machinist, and he would just make whatever we needed without any CNC machining. Another guy that really inspired me back as a PhD student was Yury Kovalchuk. He was a senior postdoc at the time. He knew everything about two-photon microscopy, and he was building an AOD scanner back in 2004/2005. It was the way he understood these systems and explained everything to me whenever I had any questions – those kind of people inspired me.

PR: From your entire academic career, what was your most rewarding project, big or small?

BP: That’s so difficult to say, because everything is kind of rewarding. What I can certainly say is that I don’t believe in putting off reward for a long time, and the idea that ‘the more you suffer, the bigger the reward’. I like it when you have smaller rewards, but more frequently.

PR: I can definitely relate to this… but at least scientific publications usually do not come so frequently. If you have to choose, which scientific publication where you took part in would you like to highlight, and what was your contribution?

BP: There was a paper in 2012 by Kamilla Angelo in Troy Margrie’s lab (paper link). I came very late to the party, all the experiments had already been done, the first version of the manuscript had been completed, and Troy just asked me to read it and give some comments. I noticed something in the analysis where the manuscript didn’t actually show unambiguously one aspect of the claim in the paper. We tried to come up with some way to design new experiments to prove that unambiguously, but at some point it occurred to me that you could just do it with the existing data, just with a different type of analysis. And once the idea had come up of doing this pair-wise scrambling of all the data points and then calculating pair-wise differences, it was very quick and easy to write some code to analyze it. And it supported exactly what we thought it would support, but now unambiguously. That felt really rewarding, to be able to nail something that would have otherwise required more experiments with a bit of clever analysis, that was really cool.

PR: Sounds like that! Especially your PI was probably really happy about this, because it saved a lot of trouble.

BP: I guess so. The paper would have been highly publishable without my input, but it was just ever so slightly better with my input; and that’s good enough for me.

“I was always more of a Malcolm Young than an Angus Young.”

PR: Why did you not become a PI yourself?

BP: Well, to start with, I was never really PI material …

PR: What would you define as PI material?

BP: Are you okay with an AC/DC reference? Would you understand what I mean when I say “I was always more of a Malcolm Young than an Angus Young”?

PR: No, not really …

BP: Malcolm Young was the rhythm guitarist in AC/DC, he was always in the background and played this steady rhythm, whereas Angus Young is the flamboyant lead guitarist in his school uniform who runs up and down the stage and is the focal point of attention for the audience. When I say “I was always more of a Malcolm Young than an Angus Young”, I’m talking about having a rock-solid line of people in the back who make sure that the lead guitarist and the lead singer can shine: I like to be in that role at the back. As a PI, you have to represent the lab, you have to present your work outside, you have to sell the ideas to funding bodies… so you have to have a little bit more of a flamboyant and outgoing nature. I’m too shy for this, so the PI path was never really on the cards for me to begin with. I was quite happy to be a sort of a medium level scientist – except that I felt there was more I could do to help other people. It felt self-indulgent to just play around with cool technology and have brainy conversations with smart people in the lab. I wanted to provide some value for others.

PR: And then you chose Scientifica. Why Scientifica? How did that happen?

BP: I did not really choose Scientifica. I wasn’t actually looking for a job when I left academia. My plan was to go back to the Bavarian Alps and work as a skiing instructor –that’s what I had done before I went to medical school. I figured that it would be really nice to do that again for a season, to pass the time until some new opportunity floated past. And then it just so happened that the opportunity to work at Scientifica floated past earlier than I had anticipated. They rang me and said, “we’ve heard you’re leaving academia and we’re looking for a software person to do some custom stuff for Scientifica’s two-photon customers”, and I was like “well, that sounds like a good idea”, and then I joined Scientifica. There was no job interview, there was no searching for a job, the job found me.

PR: How was the work like at Scientifica?

BP: Initially it was mostly intended to be customization of software, for example little Matlab add-ons for Scanimage or stuff like that. But then we also needed a resonant scanning system, and there wasn’t really any software available for resonant scanning before Vidriotech started …

PR: … and then you wrote SciScan.

BP: And that’s when I wrote SciScan. I had already written a software for resonant scanning in Labview some years before – for the frame-grabber that we discussed earlier, so I already knew what to do. While writing SciScan I also became involved in the hardware development of the resonant scanner, with the mechanical engineer and the electrical engineer. And so the job role gradually moved towards R&D management.

PR: I’m a bit younger, so I don’t know whether the resonant scanners always came from Cambridge Technology, or was there a different provider? Or did you build the scanners in-house at Scientifica?

BP: No, the resonant scanners were always the same as back in 2003 when I first started using them. The manufacturer was originally called General Scanning Inc, then it became GSI Lumonics, which later merged with Cambridge Technology, which was then bought by Novanta. There was a number of name changes involved, but no changes to the product.

PR: Have you tried out resonant scanners from other companies as well?

BP: I’m only aware of one other resonant scanner that is suitable for what we’re doing and that’s from a company called EOPC. I have not actually worked with their resonant scanners yet, but I will be in probably about two weeks from now. We have bought two of their resonant scanners, because they can be synchronized with each other.

PR: Sounds like a challenging project. Probably with two pulsed lasers?

BP: No, it’s about temporal multiplexing with a single laser. We want the scanners to run in synchrony so that we can use a single data acquisition system for the two paths.

“When I was a scientist, I was building stuff myself because there wasn’t any company that I could go to and ask them to build it for me. And when I started INSS that was still true.”

PR: Okay, this makes sense. – After Scientifica, you founded your own company, INSS. What was the biggest challenge when you got started? And why did you found your own company?

BP: The ‘why’ is very easy to explain: When I started with Scientifica, I was very close to the customers, I was building custom software modules, was interacting with scientists very closely when it came to making hardware modifications to any of the existing products or new suggestions for new hardware products. As the company grew bigger, I got more into a management role. I felt I was getting a little bit too far away from the customer and more involved in managing people and conducting things internally. It is a whole different style of working when you want to make something that is not a prototype but a production item where you want to sell 100 pieces of. Then you have to think more about questions like, how can we design it in a way that you can build it routinely and efficiently, and you have to think about all the supply chain, and build assembly instructions, and how you teach people to do that. There’s a lot of work in the background that is relatively far away from the actual experiment that the neuroscientist is going to do with that microscope and I felt like I wanted to get closer to that again. Also, if you want to sell hundreds of microscopes, it’s not really economically viable to do individual customer specials. From a business development perspective it’s perfectly understandable that Scientifica wanted to have a more standard offering and make off-the-shelf turn-key systems. But that was not so much what I was interested in. I like getting involved in changing things, not making the same thing all the time. I also felt that this sort of very customized, individualized service was not something that was available. When I was a scientist, I was building stuff myself because there wasn’t any company that I could go to and ask them to build it for me. And when I started INSS that was still true. If you’re a scientist you either buy a microscope that someone has designed to fit many purposes, but it may not be perfect for your very specific purpose. And I thought, well, surely there’s a need for modifying, customizing, making something perfect for a particular experiment and stripping out everything else that may not be needed. If you custom-build it from off-the-shelf components, you can save a lot of money. And surely from the money that people save they’d be quite happy to give me some of it, if I advise them how to do it.

PR: Didn’t it feel like shaky ground when you started? I mean, you were responsible for everything, and you didn’t get a salary anymore.

BP: I think what helped a lot is that I’ve been involved in the music scene, and I knew a lot of musicians that were self-employed. They were getting money from gigs and teaching and playing on other people’s records and they were making a living without being employed – and I felt I could do the same thing. I had been playing gigs, I knew how to ask a pub owner to pay us money for providing music in the pub. And I figured, conceptually it wouldn’t be much different whether I come to your lab and build something for you, or whether I come to your pub and play a gig for you. You just ask beforehand how much you’re going to get paid for the gig, and if it isn’t enough to cover the costs then you can’t play that gig – it’s not exactly rocket science. It was good that I’d done it for a few years in the music business, it gave me a lot more confidence that this would work out in the microscope consulting business, too.

PR: INSS is based in the UK. Have you been affected by the Brexit so far, or will you be affected by it?

BP: I haven’t been affected by Brexit yet, everything is still the same until the end of 2020. Nobody knows what sort of forms we will need to fill in next year, and what sort of new hoops we might need to jump through. I’m not going to start worrying about that now because I don’t know what it’s going to be. – Don’t get me wrong: Brexit will be disastrous on so many levels, especially now, piling onto the coronavirus crisis. And of course it’s also a little bit disappointing… I’ve lived in this country for 13 years and I feel like I made a nice contribution, doing science, paying taxes, playing music. And then all these people feel that someone like me shouldn’t be here because we’re a burden on the country and they’d rather pull up the drawbridge; and they think it’s worth taking all these economic consequences of a no-deal Brexit just to keep people like me out of their country. But if that’s what the majority voted for, then I just have to deal with this somehow. I’m reasonably optimistic that I’ll find some way to navigate these new circumstances!

PR: I get the impression that you are quite optimistic in general. Do you think this is a requirement for being self-employed?

BP: I think it’s probably a requirement if you don’t want to live a life in misery. You have to be somewhat optimistic or at least relaxed about what the future might hold because the future is kind of uncertain. You’re not going to know what’s going to happen. If you worry about it, it will not change the fact that the future is uncertain. So you might as well say “I’ll jump down that bridge when I get there”.

PR: If you don’t mind, let’s talk about some technical questions … after you set up a two-photon microscope in somebody’s lab, how do you quickly check its performance? Do you have a specific routine?

BP: The quickest performance check is to image a pollen grain slide. You check how the spiky pollen grains look like, whether you can section them nicely and how the field of view [FOV] is like. Once you’ve done it a few times, it is everything you need to get a quick qualitative idea of whether everything is working fine. If you want to do it more quantitatively then you obviously want to image beads, you want to check your field flatness, you want to get your precise FOV measurements with some grids, and so on. We have a routine that we’re working with that we constantly improve, but in the majority of cases that’s not even necessary.

PR: I think there are some companies which sell special calibration slides or samples. When would this be useful, or do you think it is overkill?

BP: I would love to use those, but they are not really suitable for 2P. You are talking about the Argolight probes?

PR: Yes…

BP: So, they specifically mention that you shouldn’t be using them with 2P, or you use them at your own risk. Also, they cost about 8’000 euros. I don’t really want to spend 8’000 euros on something where I can quickly exceed the damage threshold, and that’s why I haven’t used them. I would love to have something like that if it’s available for two photon. In the meantime, I’ll stick with these electron microscopy grids and beads and pollen grains. – What are your thoughts on those, have you tried anything like that?

PR: Several years ago I also wanted to check the field curvature for a two-photon microscope and borrowed a quite expensive calibration slide from the confocal microscopy facility, but I managed to destroy it, and that’s when I stopped experimenting. Usually, I just use beads and fluorescent plastic slides. Not so much pollen grains, because I have destroyed the slides several times.

BP: (curious) How do you destroy a pollen grain slide?

PR: Just by leaving the slide somewhere in the setup, until something makes the glass break; which happened to me twice so far. I also made some simple pollen samples in agar, but that’s not possible in winter or autumn. So I’m usually sticking to beads. But in principle I agree, pollen slides are ideal, you quickly find the samples and you can also check how the fluorescence yield changes across the FOV. Do you make your own pollen grain slides?

BP: We’re using pollen slides from Carolina Biological Supply Company. They make these ready-made pollen grain slides for schools and educational settings and the slide is like five dollars or something.

PR: I didn’t know they were so cheap! The first scanning confocal microscope that I used [a Leica SP5] had come with a pollen slide, and I had been instructed to treat the slide very carefully, because it was so expensive… So it’s good to know that it can be really cheap! – Just another question about two-photon microscopes: which preamplifier would you recommend for a standard resonant scanning two-photon microscope?

BP: We often use the Thorlabs preamps, the TIA60, and we also use the Thorlabs PMTs with the built-in preamps, which is quite convenient. You control them through USB, and you can set the amplifier bandwidth in software.

PR: I thought the bandwidth was fixed at 60 MHz?

BP: Yes, for the TIA60 preamps, it’s fixed at 60 MHz. For the PMTs with the built-in preamplifiers, you can choose between, I think, 80 MHz, 2.5 MHz and 200 kHz. When we need higher bandwidth for something, then we would use Femto preamps.

PR: Which one is your preferred preamp from Femto?

BP: I use the 400 MHz one.

“For me, cleaning always starts with these little rubber ball dust blowers.”

PR: Let’s talk about objectives… If you were postdoc in a lab and could choose any objective for testing, which one would you choose?

BP: I had a Nikon 25x NA1.1 when I was in Troy’s lab, and that’s a really nice objective. For the typical applications I was working at – population calcium imaging – that’s just a beautiful objective. I wouldn’t really need to try anything else. I’m sure there are other applications that would benefit from some of these newer Olympus long working distance objectives, but it’s so application specific, and there’s good uses for all of them. So, without having a particular experiment in mind, it would be difficult to answer the question.

PR: For these Olympus objectives, I think the working distance is something like 4 or 8 mm. Does the 8 mm make any sense for neuroscience applications?

BP: Again, it depends on your application, right? These objectives were primarily designed for cleared brains, where you don’t have much of a choice, you either use a long working distance or you’re not imaging all of it.

PR: Ok, I didn’t know that it was for designed for cleared brains. Now, this makes much more sense to me. – Speaking about objectives, how do you usually clean optical elements? Do you use tap water or distilled water, or can you recommend any other protocols?

BP: For me, cleaning always starts with these little rubber ball dust blowers. You just try to blow everything off that might be there. If that doesn’t work with the blower, you get out the compressed air can. From there, we’ll go to distilled water, and then Purosol. If I can’t get it off with that, I would go to isopropyl alcohol, and then to methanol. But we’re usually dealing with new microscopes, so in the vast majority of cases, we don’t have to go past the compressed air can.

PR: What do you do with dichroics if they’re dirty or if there’s sticky dust on top of them? Is there anything you can do?

BP: I wouldn’t really want to go far beyond the distilled water on a dichroic. If I can’t get it off with that, I might try the Purosol, but we don’t encounter dirty dichroics very often, because most of the optics we’re working with are brand new.

PR: Another question about 2P microscopes: I was always wondering about a proper way to test the alignment and performance of collection optics. How do you check whether collection optics are properly aligned?

BP: I want to capture all the light that’s coming out of the back aperture of the objective. So, if you just put some light source and a diffuser under the objective – that can be the LED on a phone with a bit of lens tissue on top as a diffuser –, you’ll get a large cone of light coming out the back aperture. Then you screw in a little target at the position where your PMT will be sitting. You should see a spot right in the center that has the size of the PMT sensor.

PR: Just to repeat, it’s important to remove the PMT in this case, right?

BP: Yes, you need to remove the PMT and put a target in its place. If you want to do it more quantitatively, it gets difficult. You could use a calibrated light source and count how many photons you get through and compare that over many microscopes and so on. But I don’t think anyone is doing that in practice.

“I don’t want to tell someone how to align a laser, I rather want to be present when they figure it out themselves.”

PR: Let’s assume a customer writes to you that there’s a problem with their two-photon system, “because the signal becomes weaker”. This is something I also often hear from less experienced colleagues or friends who ask for some help with their setup. What would be your first guess if you don’t know anything else?

BP: In the first instance I would think of it as a great opportunity to help someone to think systematically about the problem. Because if all they tell me is that “the signal becomes weaker”, then they haven’t really thought about how to communicate a problem in such a way that the other person can do something with it. I would need to know so much more , for example: what’s the signal? what’s the system? what are you measuring? have you done the same experiment over a year, and every day it gets a little bit weaker?, or is that happening within the same animal, or within the same slice? … there’s a thousand different questions. I need to find out what the person means by “signal”, what the person means by “becomes”, what they mean by “weaker”. It’ll be beneficial for that person, and for me, if I can teach them how to clarify their thoughts to themselves, and then communicate them efficiently. Maybe next time they know immediately why it’s becoming weaker simply because they’ve changed the way they think.
I have to say that I enjoy it very much to help someone to clarify to themselves what the problem is. It’s very rewarding for the person to figure out the problem by themselves, and it’s rewarding for me to teach someone a problem-solving skill that they might not have had before.

PR: I think you have also participated in some summer schools?

BP: That’s right, I’ve been at the Cold Spring Harbor imaging course five times – which is great fun, a great location and great teachers. I’m also participating every year at TENSS in Romania, the Transylvanian Experimental Neuroscience Summer School, again a great team of people that are running it with great lecturers and great spirit among students and all the TAs and lecturers. I really enjoy teaching people and helping them to figure things out. I don’t want to tell someone how to align a laser, I rather want to be present when they figure it out themselves. I might nudge them in the right direction, but if I simply told them what to do, I feel I’d be depriving them of a joyful moment of revelation.

PR: It can also be a bit dangerous to align laser beams, and especially invisible IR beams. To make the laser beam visible, do you use IR viewers?

BP: I use them occasionally, but only when I have to, so only when I’m working with a fixed-wavelength laser. And even then I try to do as much as I can with viewing cards rather than the viewer itself. Although I tend to go back and forth a little bit between card and viewer.

PR: For the tunable lasers, do you switch to visible wavelengths or what do you think about the pointing stability of those lasers?

BP: I usually align everything in the visible range and then, once we switch to IR, I image a uniform slide and check if our alignment is nice and centered. The illumination on the slide tells you whether you’ve gone off a little bit.

PR: To clarify, I was thinking more about pointing stability when changing the wavelength of the tunable laser for example from 700 nm to 900 nm…

BP: I don’t think the changes are drastic. But we’re doing the final alignment through the actual scan head based on what we’re seeing on the computer screen from the fluorescent slide, and we’re doing that at 900 or 930 nm anyway. In that sense, the visible beam is only a first approximation.

PR: But this also means that you have to be quite tall, because otherwise you cannot reach the adjustment screws of the mirrors and at the same time look at the screen to check the fluorescence of the slide…

BP: Well, you need some access to those screws and a clear visual path to the computer monitor. You can walk around the table and do it from the back, as long as you can see the computer screen, right?

PR: Yes… probably I was just thinking about some situations in the past where I tried to do this myself and needed to stretch quite far to reach the adjustment screw and at the same time look around the corner to see the screen …

BP: Have you tried moving the screen?

PR: Not really … okay, I think you’ve identified the problem, it’s just laziness from my side! – But one more question about objectives: what do you think about two-photon mesoscopes with FOVs of several millimeters? Are they more difficult to build or maintain? Do you think they will become more standard or is it just a trend?

BP: It completely depends on the application. If there are experiments that you can’t do with a standard FOV microscope, you need a wide FOV microscope. If you want to image two areas simultaneously with cellular resolution that are too far apart for a normal FOV, you don’t really have that much of a choice. A microscope like that is certainly more difficult to build, because you need custom optics for it, which are really expensive, and that is something that will prevent them from becoming really standard in every lab very soon. A lot of experiments don’t require such large FOV microscopes.

PR: So you don’t think that these objectives or lenses will become much cheaper in the future and allow them to become more standard?

BP: I don’t think they’ll go down in price very much. And they’re also difficult to maintain, and you need custom pre-chirpers. You basically need a dedicated imaging engineer in the lab who’s maintaining those microscopes, and even if you buy it commercially, for example the 2P RAM mesoscope from Thorlabs, you need someone who’s working with that microscope every day to monitor its performance and fine tune it and so on and so forth. I hope that we’ll see a few more mesoscopes popping up in the future, because there are very interesting questions that you can answer with them, and maybe the prices will come down a little bit. But I don’t think they’ll come down far enough for it to be a standard thing that everyone has.

“I wouldn’t be surprised if a three-photon microscope is on the cards at some point. We haven’t done it yet, but if someone asks, I’ll be happy to build one.”

PR: Do you also have any experience with setting up confocal microscopes or light-sheet microscopes and, if yes, how is this different from setting up two-photon microscopes?

BP: I have not set up a confocal microscope so far, but I have set up a mesoSPIM light sheet microscope that your colleague Fabian [Voigt] in Fritjof [Helmchen]’s lab has designed. It was a very different experience in the sense that this was probably the first time that we’ve built something where we had basically no input in the design, and we were following someone else’s instructions. That was a little bit daunting, simply because I’d never done it before, but Fabian’s documentation is just so excellent that it turned out to be not a problem at all, and in that sense it was not much different to what we usually do when building a two-photon.

PR: Do you also have any plans of building a three-photon microscope?

BP: I’m going to build whatever people ask me to build. We’ve done a lot of 2p microscopes, but we’ve also built wide-field microscopes, we’ve built a mesoSPIM, we’re working on a microscope for in-situ sequencing, I’ve helped people with LabView coding. Whatever it is that people ask me, if I have time to do it and it sounds interesting, I’ll do it. And I wouldn’t be surprised if a three-photon microscope is on the cards at some point. We haven’t done it yet, but if someone asks, I’ll be happy to build one.

PR: So, even if I gave you a lot of money to make you build a 3D AOD-based two-photon scanning microscope, would you give it a try? I’ve heard that it is extremely challenging to make this run stably.

BP: I would certainly try it – and get the right people involved. I was actually a co-author on an AOD paper some ten years ago with people from Angus Silver’s lab. AOD systems are a little bit finicky, but with the right person, with the right project, with the right collaborators on board, I don’t see why someone who wants an AOD system shouldn’t attempt to build one. I’m sure that some of these teething problems that any new technology always has will be ironed out over the next few years

PR: So you’re quite optimistic about the future role of AOD scopes for two-photon microscopy?

BP: For the right applications – yes.

PR: As we mentioned you also provide your service in order to help your customers save costs. If a lab really wants to save money, would you recommend to buy a fixed-wavelength laser for two-photon microscopy? I’m also not sure whether there are already some 920 nanometer lasers around …

BP: There are now a few manufacturers that build fixed wavelength 920 nanometer lasers, and if someone says, all I’m going to do with this microscope for the next three or four years is GCaMP imaging and I just want to have a workhorse for that, then that’s certainly a good option. If it’s a young lab that only has a single two-photon microscope in the lab, and they don’t know exactly what sort of projects might come along next year and what sort of dyes they might be using next year, then it’s a bit of a risk to bet all your money on a single wavelength. This is probably more something for labs that have another two-photon with a tunable laser, where they can do experiments in the red or where they can explore other dyes that aren’t excitable at 920 nm.

PR: Do you have examples for companies which sell fixed wavelength lasers at 920 nm?

BP: Coherent are selling one now, the Axon 920; Spark Lasers have a few options for 920 nm; there’s Menlo, a company in Munich that sells the Ylmo-930; Toptica have a 920 model, also NKT Photonics – there’s quite a few around now. I haven’t actually tried any of them yet, but it’s certainly going in a direction where we’ll be seeing these more often in the field.

PR: Another technical question – you mentioned it already: what’s your opinion on multiplexed acquisition? Will it remain a niche product like the mesoscopes, or do you think it will become much more standard?

BP: Again it depends completely on your application … if you want to image two areas simultaneously without having to jump between them – whether that’s on a mesoscope where you image two different brain areas that are millimeters apart, or whether you want to image a single neuron and you want to image soma and spines both with very zoomed-in resolutions, but without having to jump between them – then multiplexed acquisition can be the right technique. If you don’t have the right question for multiplexed acquisition, then there’s no point getting it. All of these things are only useful in a very particular context that depends on the scientific question that you want to answer.

PR: I always thought that multiplexed acquisition might be particularly difficult to implement because it’s not only challenging in terms of hardware and optical alignment, but also the software to disentangle the two multiplexed photon streams is not trivial. I was thinking that this combination of both hardware and software challenges might be a bottleneck which prevents many people from doing it?

BP: Well… you can also de-multiplex the channels in hardware using an analog system. Spencer Smith and Jeffrey Stirman used a nice system in their 2016 Nature Biotechnology paper with a wide FOV (paper link), where they actually had an analog photon-counting system and then just acquired with a standard data acquisition system. They didn’t need to change anything in software because all the photon counting was done in the analog domain. But now, with these gigahertz data acquisition systems, you can acquire the data fast enough, get the laser timing and then assign different acquisition time bins to different channels. Scanimage can do that now, so it’s not really a challenge any more.

PR: Do you have an opinion about the new vDAQ system of Scanimage? I think it’s not based on National Instruments hardware any more.

BP: We’ve built a couple of microscopes with those systems, and they seem very nice, very user-friendly. There’s no obvious downside, although I haven’t had a chance to really observe them over the same time periods as I have with National Instruments for over 20 years. But I have very high hopes for these systems, and we’re using them as standard now. The only situation where we’re not using them at the moment is when we have to count photons, because the data acquisition speed is currently 125 MHz max on the vDAQ. But there will be a new vDAQ version coming out later this year, with high-speed acquisition that will then be suitable for photon counting.

“I had to fly to Okinawa to build a microscope, but I didn’t know whether my toolkit would be there, too.”

PR: I see. Maybe, to finish up, some fun questions… or maybe not so much fun, let’s see. During your time at INSS, what was your most challenging project?

BP: One of the first microscopes we built was in Okinawa, Japan. Shipping all the microscope parts to Okinawa was a bit of a challenge. Three of the four boxes arrived without problem, but the box with my toolkit didn’t arrive and was completely lost for about a week or so. UPS didn’t know where it was. They had scanned it in Tokyo, but then lost track of it and had absolutely no idea where it was. I had to fly to Okinawa to build a microscope, but I didn’t know whether my toolkit would be there, too. I had lots of spare parts in the toolkit, and all sorts of specialist tools that were difficult to replace in the shortness of time. That was a challenge to my otherwise unshakable optimism … In the end, the toolkit did arrive and everything was fine; I built the microscope, no problem with that. But then I needed to ship the toolkit back, and UPS kept cancelling the booking and no one on the customer service hotline knew why. We eventually shipped it back with Fedex. It was all very annoying, and I then decided to get a shipping agent who’s now managing all of our shipping for us, and he’s been brilliant.

PR: Okay, this sounds like a rough start…

BP: Yes … having these useful conversations with UPS customer service … It makes my blood boil just thinking about it.

PR: What was the most expensive device you destroyed by accident?

BP: I don’t really know, I don’t remember.

PR: You never destroyed an objective?

BP: I never destroyed an objective, never destroyed a laser; I’ve destroyed scan mirrors, ETLs, stuff like that, probably a few other items in that price range. But I move on from that pretty quickly. I always think of it as a tuition fee that I have to pay, and then I forget about the incident. But the knowledge of what not to do stays with me.

PR: Which skill do you wish you have learned earlier that would be of use for your current job?

BP: Hm, all of them! I would love to have university degrees in electronic engineering and optical physics and project management, computer science, business administration, and applied math. Almost every technical thing or business thing that you could learn at a university would be in some way useful to my current job. But I also have to be realistic about how much I can do with the available time. These disciplines have crossed my path sort of superficially, and I’ve picked up what I can. I wish I had a really solid foundation in all of them, but I have to live with my limitations.

PR: This sounds quite modest, given your expertise in so many domains… if you had to pick one, what would you learn if you had one year for that?

BP: Probably math. I’d really like to go back to undergraduate level math and learn all the foundations properly.

PR: From your long experience in research and as a consultant, what kind of advice would you give to students or postdocs who want to learn the skills which are needed to understand a microscope or, more generally, to set up experiments?

BP: You just have to start, just go and do it. If you want to set up a microscope, go online, google “how to set up a microscope”, and then click your way to the websites where you can find how to set up a microscope. Maybe someone recommends to go to a course, maybe someone recommends a particular Youtube video. You’ll find all the resources that you need. This is such a great time to be alive, because you can find everything that you need online. You can apply for courses like the CSHL course that we discussed about, or TENSS, or numerous other imaging courses. If you don’t get into any of those courses, you can still learn it all online and just start building. A lot of people now offer very simple 3D-printed parts to set up basic optics and teach yourself the skills. You don’t even have to wait until you’re a student or a postdoc, you can do that as a little school project when you’re 12 years old, because all the learning resources are online.

“I would recommend to stop thinking about a career. I would recommend to think about just getting better and better at whatever it is that floats your boat.”

PR: So you would encourage self-learning, because that’s the way to learn what you have learned yourself?

BP: It’s one way that works for me. Other people have different working styles, and your own working style emerges when you start doing something.

PR: What would be your advice for young scientists like PhD candidates or postdocs who think about a career like yours?

BP: I would recommend to stop thinking about a career. I would recommend to think about just getting better and better at whatever it is that floats your boat; if you work more like an artist or an artisan and just focus on improving whatever your chosen art or your chosen craft is, then you’re going to get good at it, and then you’ll enjoy doing it. And if you keep an open mind, intriguing opportunities will pop up. And if you pursue these opportunities whenever they present themselves, then you’ll end up with the very tortuous career trajectory that I have, that doesn’t seem to make any obvious sense from outside. But for me, internally, it had a very natural flow. You’re obviously also going to need a massive amount of good luck; but I don’t really have any advice on how to get lucky.

On a side note, for anyone who’s considering starting their own business or doing something on their own, there’s an old blog post written by a banjo player called Danny Barnes. He writes about how to make a living by playing music (, and while the blog post is ostensibly about music, there’s a lot of advice that applies to any self-employed person, not just musicians; and also not just to self-employed people, it applies to anyone who is in some way in a largely self-directed job – which I guess is the case for most scientists. There are a few paragraphs in there that are more specific to music, but I think it’s just a very good read on how you want to conduct business and life with other people and make a living from it.

PR: I’m curious, and I’ll check it out! … I think it has become obvious by now that you’re a big music aficionado. On Twitter, you describe yourself, among others, as ‘Bluegrass Dobroist’. Could you explain this to somebody without any clue, like myself?

BP: A Dobro is an American instrument, it’s like an acoustic guitar that has an aluminium resonator cone built into the body. Originally, it was invented to make a normal guitar louder, a bit like a speaker cone that picks up the vibrations from the strings and projects them outward. The Dobro was invented in the 1920s to make the guitar a little bit louder to be heard over orchestras, and it was kind of popular for a few years, until someone invented an electric guitar that you could just very easily make a lot louder, and then the Dobro fell into obscurity. It’s played in a lap style, and you’re not fretting the strings with your fingers like on a normal guitar, but you use a steel bar that slides up and down the strings.

(Since I did not fully understand the following explanations, he pulls out a Dobro and simply shows to me via video how it works to play a Dobro.)

PR: And what is the meaning of ‘bluegrass’?

BP: Bluegrass originally comes from the American state of Kentucky. Kentucky has a certain type of grass that has a sort of a blue appearance, and that’s why Kentucky is called the bluegrass state. There was a band in the 1940s that came from Kentucky; the band leader was called Bill Monroe and this band was called the Bluegrass Boys. Bill Monroe and the Bluegrass Boys played a very new and unique style of acoustic country music, and then other people started copying that style, and eventually because of the name of the band the style became known as Bluegrass music, and that evolved into a whole genre of music. Now, there’s tons of bluegrass bands, and bluegrass music, bluegrass festivals, and there’s a Grammy for bluegrass music. So, from this one band in the 1940s a very huge branch of music has emerged.

Bruno Pichler and the Hot Rock Pilgrims.

PR: Interesting, I didn’t know that.

BP: I’ll be most happy to talk a lot more about bluegrass music all day long! I love it – I’ve been really involved in this scene for the last 12 years or so, going to festivals almost every weekend – obviously not now with the pandemic, and generally a little less since I started the company and since we had a baby two years ago. I’ve got a lot of other things to do now, but I’m still very much connected to the whole music scene and to the guys in the band and all that. Sometimes during the time when I worked at Scientifica, I was playing five, six, seven gigs a month even though I was living here in East Sussex and drove an hour and a half or two hours into London after work to play gigs and come back during the night to be back at Scientifica in the morning…

PR: At the end, is there anything else you want to share?

BP: I want to give a quick shout-out to my team, Angelos, Eleanor, Mark and Simon, who are just a massive pleasure to work with.
Other than that, if people want any sort of clarification on the things that I’ve mentioned, if people are thinking about transitioning from academia to industry and there are questions popping up, send me an email or hit me up on twitter and I’ll be quite happy to answer questions and give advice!

PR: Thank you. I think you gave already a lot of good advice here!

BP: I’m glad to hear that.

PR: Thank you very much.

(c) Bruno Pichler
This entry was posted in Calcium Imaging, Data analysis, Imaging, Microscopy and tagged , , . Bookmark the permalink.

1 Response to Interview with Bruno Pichler

  1. Sias Jordaan says:

    Nice interview – Good luck Dr Bluegrass!

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