Wavelengths Spring 2016 Interview Dr. Enrico Gratton, Founder and Principal Investigator, Laboratory for Fluorescence Dynamics at UC Irvine We delved into Dr. Enrico Gratton’s lifelong quest to develop new fluorescence techniques for unlocking the mysteries of complex biomolecules and their behavior in cells. Semrock: Your doctorate degree was in Physics, yet your thesis was on the acid denaturation of DNA – an unusual topic for a physicist. At that time, interdisciplinary science was quite new, was it not? Dr. Gratton: My thesis was all about the study of single molecules, which was very unusual at the time, but the work I began there was something I continued for the rest of my life. It is physics in some ways, but it is also biophysics. I always wanted to do biophysics, but always from the physics point of view. At that time, people would say, “What is biophysics? Is it physics? Is it biology?” At that time, there was a lot of discussion about this, and there were no courses in biophysics. A study of single biomolecules isolated from cells was something that could come only from physics – a biologist would dismiss it, saying, “This is not something that comes from nature.” When I started biophysics, the genetic code had just been published. It was a new era – we needed to understand what the alphabet was. After 40 years, we can decipher the entire human genome. The progress has been enormous, mainly due to development of fluorescence methods originally used in the genome project. Semrock: What advice do you give to newly graduating students looking to choose a field in which to study for their PhD in this day and age? Dr. Gratton: In my lab, we have two ways students begin their research. One is to develop instrumentation, and another is to apply that instrumentation to entirely new problems in biology. So it depends on whether people are interested in the engineering or the biological part. We have interesting work in every area, and I believe that in everything you do, you will always find something interesting. Semrock: What drew you to study cell structure and behavior? Dr. Gratton: The study of single molecules depends very much on the methods, and I find these very interesting. The statistical techniques are important – to see the signal within the noise and understand what is happening. A physicist takes a very different point of view to this problem, one of looking at what generates this noise and how to see beyond it. I have always worked in methods and techniques, developing new ways to analyze the data and then applying it to problems. Semrock: How did the availability of our hard-coated optical filters with steep edges and deep blocking affect your research? Dr. Gratton: I have been a Semrock customer for many years, since the company started, and these kinds of filters have allowed us to do much better things. Narrow filters with 98% transmission were crucial to the development of fluorescence correlation spectroscopy (FCS) techniques. The correlation function scales as the square of the signal, so better filters enabled the measurement to be possible. Narrow filters with steep edges have also been very useful for multiphoton fluorescence to avoid the detector being saturated by the excitation laser. For this, we needed out of band blocking that was very, very high to avoid being overwhelmed by the laser reflection. These filters have made possible some of the technologies that otherwise we would not have today. And still I get very good customer response from Semrock – you are willing to discuss and do unusual things. Semrock: You use fluorescence fluctuation analysis frequently when studying proteins. Why is this technique well suited to the movement of these structures? Does optical filter performance play a role in the success of that analysis? Dr. Gratton: We use fluorescence fluctuation analysis in order to measure the number of molecules. When signals are very small, you have to capture these signals very carefully, and it is important to have a good filter. Semrock: You’ve used Semrock filters in the development of your IC 3D system for detection of low-abundance biomarkers in biological samples. Can you tell us a little bit more about the technology? Dr. Gratton: This instrument is used for analysis of bacterial infection in the bloodstream. The idea is something that comes from the single molecule experiments I did many years ago. Someone asked if I could distinguish single bacteria, and I said of course – and so it was. By scaling the system for single molecules and using droplet microencapsulation to bind our DNAzyme sensor to bacteria coming from unprocessed blood, you can see very few bacterial particles from within a billion droplets. Editor’s note: The IC 3D system, currently under commercialization, has the potential to improve outcomes for patients suffering from antibiotic-resistant pathogens through early detection. Learn more about the technology. Semrock: Time-resolved fluorescence (TRF) spectroscopy seems to be another popular technique in your lab to study movement and dynamic changes in cells. What secrets can TRF spectroscopy reveal for the diagnosis and treatment of diseases like Huntington’s, kidney disorders, and cancers? Dr. Gratton: It is very much in the technique. We can do experiments that other people can find very difficult – for example Huntington’s disease – we looked at protein aggregation while it is occurring, which may be an important part of the disease. Formation, size, location of these very large aggregates is possible to observe using this technique. Editor’s note: This research used a number and brightness (N&B) method to measure the molecular brightness of the protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. Read the original article in Biophysical Journal. Semrock: You’ve done some work with calibrated, FRET-based tension sensors. How can these tools be used to better understand biological systems? Dr. Gratton: Those sensors were used to look at forces between cells. Cells are held together by contacts, and we wanted to be able to measure the forces between cells, across cells. How to measure this and be relatively precise required new techniques. Looking at changes in lifetime of the donor and acceptor made this possible. Semrock: We were intrigued by a study in which you applied selective plane illumination microscopy (SPIM) to 3D fluorescence anisotropy imaging of live cells. Can you tell us more about why you took this approach? Dr. Gratton: The reason is that in the SPIM microscope you have two objectives, much as when you use a standard a fluorimeter. So everything that we do with a fluorimeter, we can do now with a microscope, except in a very small volume. Everything that we know how to do with fluorescence anisotropy we can do now in microscopy. In this paper, we measure FRET homotransfer in proteins. In some way, this is easier than using time resolved for FRET in the SPIM microscope. You cannot use ratios of intensities, so anisotropy is the only way to measure the interactions of these proteins – this is the only way possible. Semrock: How do you think the remaining mysteries of the cell can be unlocked? Dr. Gratton: Trying to look at each molecule and how it interacts with other molecules – what is its life within the cell – this is a biophysical problem. Cells have spontaneous fluorescence and it is important to understand what those molecules are and what changes in autofluorescence are telling us. When you want to study something that arises from human tissues, looking at what nature has given us in terms of signal can be very useful – markers for the metabolic state of the cell, stress on the cells – these are things that can be very important for various kinds of studies in cells and small laboratory animals. Semrock: What do you think will be the most active area in your research in 5 years? Dr. Gratton: Fluctuations are very similar to the techniques of super resolution microscopy (SRM), which are based on the blinking of molecules. I think there is the possibility to combine these two. One of the problems with SRM measurements is that they take time to collect, so you lose most of the dynamic information. I believe there is the chance to capture the information of both techniques, including the dynamics. We will work in the coming years to see if this can be done. Semrock: Is there anything else you would like to share with our readers? Dr. Gratton: Our lab is open to public for people to come and do experiments. We are always looking for new and interesting applications. We can give people access to these new technologies – everything we develop is available to people – and we welcome these collaborations. Visit the Laboratory for Fluorescence Dynamics webpage. Semrock: Thank you very much for your time, Dr. Gratton, and for sharing your work in developing new techniques to look at cellular biophysics through fluorescence.