The Role of Microscopy in Improving the Human Condition

Thank you for the opportunity to be with you today. The optical microscope was developed early in the 17th century and, after 50 years of improvement, microscopists such as Hooke and Van Leeuwenhoek were opening up an entire new micro world of living systems, beyond the ability of what the naked eye could see. Microscopes steadily improved until the end of the 19th Century through, oftentimes, with fits and starts and a lack of reproducibility. Then, in the 19th century, a microscope manufacturer named Carl Zeiss teamed up with physicist Ernst Abbe and chemist Otto Schott to make a science of microscopy. Together they figured out exactly how images are formed in the microscope and how to create lenses to optimize these. One of the consequences of their work was that Abbe discovered that there was a fundamental limit to the smallest object you could resolve in an optical microscope, which was half the wavelength of light. Because they were able to make a reproducible scientific instrument, optical microscopy became ubiquitous in the 20th Century. In fact, it is so ubiquitous today that if you did the deletion test and erased optical microscopes, there’s no branch of science and technology that would not be impacted by their absence. However, because they attained the predicted resolution of the microscope at the end of the 19th Century, in some ways the technology stagnated, so that the microscope you could buy from Zeiss in 1880 was really not all that different from the microscope you could buy from them in 1980. There were additional contrast mechanisms and better light sources and such, but it was basically the same instrument.

As a result, in the 20th Century, it was the developing fields of Biochemistry and Molecular Biology that became predominant. In fact, they’ve been so successful that today most biologists have a worldview where they look at living systems through the eyes of biochemistry or molecular biology. But that started to change, starting around 1980, thanks to a series of unrelated technical innovations. The first was the transistor, which led to the personal computer, so that you could control microscopes automatically. It also led to semiconductor cameras that could detect light very efficiently in digital images, and these could be processed and analyzed by computers. The next innovation was immunofluorescence technologies where, instead of seeing ghost-like images in black and white of cells, you could illuminate any one of the tens of thousands of different types of proteins specifically, and see them individually. That had another big boost in the 90s, with the development of fluorescent proteins, where you could coax live cells to produce their own fluorescent labels. The third was a development of lasers, which allowed us to illuminate these fluorophores very precisely and sculpt their light in different ways in space and time to create new microscopes.

As a result, by stirring these technologies together in different ways, there has been a Cambrian explosion of microscopy technology since the 80s, first with the confocal microscope, then spinning disk confocal microscopes that look at live cells, two-photon microscopes that look in scattering tissue, super-resolution microscopes, like the ones I developed, that see beyond Abbe’s limit, light sheet microscopes that can look at 3D dynamics, and adaptive optical microscopes that can look into aberrating multicellular tissues.

What these microscopes have revealed is that the cell is incredibly complex and incredibly dynamic and, if you don’t take into consideration the fact that it’s the dynamism that defines life, you are missing a critically important aspect of what’s going on.

I’ll give you a couple examples of that. First (Figure 1A), using a super resolution technique that I had a hand in developing, one of our collaborators looked at the Huntingtin protein, which is involved in Huntington’s disease. Here you see the nuclei of two live cells. The one on the left with the normal or “wild-type” form of the Huntington protein, creates only very small, intransient aggregates, but when you have the glutamine repeat of the mutation in the disease, you get these large aggregates in the nucleus.

Now, people have known about these aggregates for a long time, but by imaging in a second color our collaborators were able to show that individual transcription factor molecules get bound up in within the aggregates like they’re caught in quicksand. This suggests a previously unconsidered possibility that the capturing of these molecules may lead downstream to the underproduction of certain important proteins critical to normal cell metabolism, because the transcription factors can never get to the DNA to produce the necessary RNA to produce these proteins. Thus, a new possible avenue of attack on the disease was found that would be hard to discover without the sort of direct imaging done here.

In a second example (Figure 1B), the textbook picture of a cell shows its key components, or organelles, such as its mitochondria and endoplasmic reticulum (ER), as independent bodies floating passively within the cytoplasm. The reality is not like that at all. Using a high-speed super-resolution method known as structured illumination microscopy, we found that the microtubule cytoskeleton constantly remodels the ER, sometimes under the influence of protein-degrading lysosomes that track along the microtubules like railway cars and push the ER around. Similarly, when we looked at a cell with six labeled organelles using yet another microscope that excels at imaging dynamics in three dimensions, we found that contacts between organelles are ubiquitous. In particular, contacts between mitochondria and the endoplasmic reticulum act as organizing centers for other organelles to come and dock temporarily for biochemical reactions. So the cell isn’t a bunch of individual isolated components, but rather a bunch of constantly interacting components with transient interactions happening everywhere which are essential to its function.

The problem with the techniques I’ve shown so far is that they look at cells in isolation. However, biologists know that the appearance, or phenotypes, that we observe are the result of gene expression, and gene expression is influenced by the local environment. Therefore, when we look at cells on a coverslip, we’re far removed from the multicellular environment where they evolved, and we can expect them to look and act different than they do in whole organisms. In order to see them in their true physiological, multicellular context, we’ve borrowed the technique of adaptive optics from astronomers, which we use to cancel the optical aberrations present in multicellular systems to be able to study their dynamism in vivo with full, unperturbed resolution. For example, we can follow the complex movements of an immune cell as it migrates through the inner ear of a fish. In your body you have 37 trillion cells all undergoing that type of choreography at the same time. Furthermore, inside each one of those cells, several billion protein molecules are stochastically buzzing around and interacting in a way similar to that I showed in the Huntington’s example earlier. Inside each human body there is an entire universe of complexity, and inside the collective brains of all of humanity there are more synapses than there are stars in the universe. The most complex matter in the known universe is us.

The upshot is that we’ve made remarkable progress in the past thirty years in developing microscopes that allow us to see and understand living systems in ways we’ve never been able to before. The bad news is that it remains a herculean task to make these microscopes accessible. That’s true not only for the advanced microscopes I’ve introduced here, but also the simplest microscopes that have been around for decades – microscopes that could have an impact in third world countries, particularly for the diagnosis and treatment of infectious diseases.

Here (Figure 2) you see examples of some of these diseases, which afflict many millions of people every year. Often you can identify them directly by the outward phenotypic changes they induce to the appearance of the afflicted, but if you want to understand what specific infective strains are involved, the degree of infectious progression, and the efficacy of treatment, access to basic microscopy would be a huge help. That’s routine in developed countries with the extensive resources they have. Several groups, particularly over the last decade, have taken on the challenge of making extremely inexpensive microscopes to make them broadly accessible in the third world. The Foldscope, for example, from Manu Prakash at Stanford, is less than a dollar. It’s very much like Van Leeuwenhoek’s old microscope hundreds of years ago. Nevertheless, there are many problems other than just cost that have limited the impact of such efforts, such as surrounding technical infrastructure, training, and resistance to seek treatment. I think these problems are solvable, but they require a concerted effort by a number of different communities.

At the other end of the technological spectrum, we have an analogous problem with the adoption of the microscopes we make. While they’re very powerful and can routinely reveal biological processes never seen before, they’re also very complex and expensive. We want them to be in the hands of many biologists, but there are very few optical engineers in the world who can build them, and the overlap between these two groups is even smaller. With our lattice light sheet microscope, we’ve worked very hard to try disseminate the technology to others, but while we’ve sent detailed plans to ~90 groups and offer significant support on top of that, so far only ~ 20 are operational in four years of effort. Ultimately, the only way biologists are going to broadly adopt these technologies is if they can be commercialized and made turnkey. You heard from Helen Blau’s talk that biologists have many, many other things to worry about than whether their microscope is working properly, so they need something that is easy to use and works routinely. To do that, you need market forces to provide the incentive to invest in the necessary product development and thereby cross the common valley of death between a research idea and a commercialized product. This usually becomes a chicken-and-egg problem, because companies aren’t going to invest the money to make these microscopes until they know there’s a sizable market for them, and biologists won’t tell them there’s a market until there’s a useful microscope for them to work with. To get around this problem, at Janelia we founded an Advanced Imaging Center where we have a suite of pre-commercial microscopes we’ve developed, which we offer free for use to visitors from around the world. This has been very successful, and in a couple of instances it’s been enough to get a couple of these microscope technologies into the commercial pipeline.

Beyond that, we face other problems as microscope developers. Just as you can’t build a car that can take the kids to daycare on Friday and then win at the Indianapolis Speedway on Sunday, you can’t build one microscope that can do everything. Microscopes have to differentiate. That’s a problem, because, if you have an imaging core for biologists, that means they need to have many microscopes of different types there and they don’t know which one the biologists are going to need on any given day. As a result, most of them are underutilized. The final project I’ve been working on at Janelia is to make a Swiss Army knife microscope that takes almost all of those technologies I’ve told you about, and puts them in one four-foot by four-foot box. We worked hard to design the microscope to be affordable and exhaustively documented and, while initially it will again only attract the small subset of people with the skills to both build it and use it, hopefully the ideas within it will eventually hit the commercial sphere. At that point, imaging cores could have a suite of identical microscopes, where the operator could push a button to reconfigure the system to whatever mode of microscopy works best for any particular biological user.

Yet another problem created by our microscopes is that they can produce terabytes of data per hour. We have one paper that was just accepted that includes nearly a petabyte (one million gigabytes) of data. Most scientists do not have access to the computational resources needed to extract biological meaning from data at this scale. You can create beautiful movies of animated cells, but you’re not going to gain a detailed understanding of transient interactions happening across the cell over a wide range of spatial and temporal scales without massive computational resources. I’ve recently moved to Berkeley and we’re trying to attack this problem with a new Advanced Bioimaging Center, modeled after the Center we founded at Janelia, but with a much greater emphasis on developing open-source algorithms so that scientists can get quantitative biological insights from the massive data sets these microscopes produce.

To conclude, as a physicist I come to biology as an outsider. Modern biology reminds me of the old parable of the blind men and the elephant: when you’re trying to understand living things, the molecular biologists are holding the tail, the biochemists are holding the trunk and perceive something different, and the structural biologists are hanging onto an ear and sense a third thing. The thing that defines life is that it’s animate, so the only way we’re really going to understand life is to understand dynamic interactions, which is what modern optical microscopy brings to the table. It may never reveal the whole elephant, but at least we can hold another part of it to understand biology in a new way.

Thank you.