Building the Power Tools of Science

NIBIB’s Intramural Labs Team Up to
Explore the Nano Universe

For many years, for whatever reason, science has stood quietly by, tolerating jabs about its not-so-cool, dare we suggest, “nerdy” status. But the buzz words used to describe the science in NIBIB’s High Resolution Optical Imaging (HROI) and Biophotonics Sections—glowing green mutant proteins, super resolution, laser beams, nano 3-D—conjure mental images more befitting a popular video game than blasé bench top science. Further inquiry about the imaging tools being built, and the ultimate vision of the investigators concentrated on the effort—namely Drs. George Patterson and Hari Shroff—reveals a story that certainly bucks all conventional stereotypes.

The Hubble Telescope Nano Style

Just as the Hubble Telescope enables researchers to see and explore much greater expanses of the universe with breathtaking clarity, in the High Resolution Optical Imaging lab, Dr. Hari Shroff is developing microscope techniques that provide clearer pictures of nano-scale structures and processes on the surface and inside single living cells. His primary challenge: breaking the diffraction barrier.

Shroff explains, “We are developing new super resolution tools and techniques that will allow us to see things that are much smaller than a single cell. When I say ‘super resolution,’ I am talking about trying to get clear pictures of objects that are even much smaller than the physical size of a wavelength of light—this is what we call the diffraction limit. Microscopes, like everyday cameras, yield images constructed from light. So this physical property of light itself is a significant obstacle to current studies in cell biology because so many of the things we are now trying to explore are at the nano level.”

Top Row: Dr. Andrew York, HROI Research Fellow Second row: (left) Dr. George Patterson, Biophotonics lab Chief; (right) Dr. Hari Shroff, HROI lab Chief Lower row: (left) Ali Ghitani, HROI Post-bac Intramural Research Training Award recipient, (middle) Dr. Yan Fu, Biophotonics Visiting Fellow; (right)  ORWH-NIH-FAES summer program participant, Monique McCants   Photos and story by Jude Gustafson

Microscopy methods employ fluorescent dyes that are programmed to attach to specific objects of interest inside cells. Making a comparison to what one might see, or might not see, when a pea is suspended directly in the front and center of a 60-inch, intensely bright searchlight, Shroff says, “A fluorescing dye will attach itself to the object we want to look at, but when it is stimulated enough to fluoresce, the light wave it produces is about 250 times larger than the actual object we’re trying to see. Subsequently, the resulting image is a blurry blob of light. So the central problem for microscopy is figuring out the true dimension and location of the object through this blur.”

So why not use smaller wavelengths? First of all, shorter wavelength sources—from deep UV to x-ray—are not widely available, but even if they were, not many dyes fluoresce when stimulated by these wavelengths. Secondly, the shorter the wavelength, the more potential there is for damage to the cell or its contents.

Shroff continues, “Let’s say you have one molecule that you’re interested in that is one nanometer in size, or in other words, one billionth of a meter in size. What you see with a microscope is 250 to 300 times bigger than that molecule, so if you have many of these molecules inside a cell, the comparatively large light waves distort and hide the true location and distribution of the objects.”

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Hollywood and the High Resolution Optical Imaging Lab

Fans of any current-day television crime series have seen the blurry crime scene photo magically and quickly made clear enough for positive, unmistakable identification of the suspect. Shroff says, “What we’re trying to do aims for the same kind of clear image results, but the process we use to get a clearer image is not the same.”

“In the nano world, it doesn’t matter how large you might blow a microscope image up. There would still be blur because light wavelengths are so much larger than the object you’re trying to see. But we’re now able to look beneath that level of blur using a technique called Photo-activated Localization Microscopy (PALM), a method developed and first demonstrated at the NIH by Eric Betzig and Harald Hess.” 

Turn it Down!

Shroff’s post-doctoral work on PALM utilizes basic concepts of its forerunners, but modifications of this method set it a decided pace ahead with impressive improvements in image clarity and resolution.

“Let’s imagine that I have attached some green fluorescent proteins (GFP) to some proteins I want to know more about inside a cell. If I make all the proteins fluoresce at once, I can’t know where those cell proteins are because they’re lost in a blur of light. But if only a few of the protein molecules fluoresce at a time—and ideally, those few should be well-separated in space—I could locate their centers very accurately, and that’s where the nano-sized cell proteins are that I want to study. I continue my protein hunt by turning on other GFPs—just a few at a time—sequentially plotting the center of each, one-by-one, which gives me a kind of map that much more precisely shows where my proteins of interest are in the cell, as well as where they are in relation to each other. If you only plot the centers, then you get this much more highly resolved image.”

Digging into the details a little further, Shroff adds, “There is a concept called ‘activation’ that I use like a light switch to turn neighboring molecules off and on. It’s kind of like a pinball machine; each activation involves one photon, which is a packet of light energy. Photons must be absorbed by a dye molecule in order for that molecule to become activated and ready to shine. In most photoactivation experiments, a sample is barraged with photons to ensure the activation signal is strong enough. In the case of our PALM studies, however, a much lower laser intensity, where only a few molecules are activated at a time, is optimal.”

Capitalizing on a natural chemical reaction seen in a glowing jelly fish species named Aequorea victoria, Dr. George Patterson used the creature’s bioluminescent gene to invent the first photoactivatible GFP in 2002. His invention is now part of an ever-expanding rainbow of nano-enabling fluorescent tools that go hand-in-hand with PALM microscopy.

Shroff says, “I am in the microscope business, so what George does is very relevant for me because the fluorescent markers that his lab is developing could provide even higher resolution views of various cell structures. In essence, if you can tag it with a photoactivatable fluorescent molecule, you can image it much better using this technique.”

So What?!?

The ability to see the nano-sized world with such clarity is truly marvelous, but what does all this imply in terms of human health?

Shroff explains, “There is only so much that you can infer from conventional microscopy images, but if you can see more clearly, then perhaps someday you can figure out what’s going wrong and target that in terms of intervention. We’re digging further down with questions whose answers are hopefully closer now because of this technology. For example, what takes place inside a cell as it divides into two daughter cells, and what initiates and makes that process happen? Are there ways we could inhibit cellular division when it goes awry, like in the case of cancer? Is there some way we could make cells grow and migrate properly for healing of paralysis or brain injury? How do proteins within a cell interact to give rise to function, or perhaps more importantly, give rise to malfunction and illness? We have answered so many questions with conventional microscopes, and similarly, although we have a long way to go in development, this technology has the potential to expand our knowledge all the more.”

Shroff points out quite matter-of-factly that cells are not two dimensional, “Our research is now pushing out into a third dimension. What we’ve done so far is great for studying proteins that cluster at the cell surface, but we want to accurately image the cell nucleus and other internal structures and processes.”

In another room, across the hall, brightly colored lasers cast an “other-worldly” glow on HROI team member, Dr. Andrew York, who is busy developing a recipe for a new 3-D microscope. Applying confined “sheets” of light to sequential planes of a worm embryo, the device collects slices of data that will be reconstructed into a 3-D image in similar fashion to MRI technology.

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Learning from Worms

Shroff uses C. elegans, a tiny worm found far and wide in soil, for studies on neural development. He walks to an incubator and pulls out a Petri dish. Removing the lid, he reveals what most would guess to be tiny specs of dust. The boyish enthusiasm in his voice makes it seem as though he is talking about a favorite pet. “They’re a really easy organism to grow and maintain, because you can feed them E. coli and they self-reproduce. You can hardly see the adults with your eyes because they’re thinner than a hair and only about a millimeter long, but the main reason these are good for our microscopy studies is because they’re transparent. You can see right through them.”

C. elegans embryos, the primary subjects of Shroff’s studies, are about 30 to 50 microns in length, which is too small to see with the naked eye, but large enough to be seen in a standard microscope. For many reasons, they have become popular in research, and thus, are a well-studied model organism. The species' genome has been sequenced, so there is considerable control over genetic factors, and with only about 1,000 cells in the mature adult, identity of all its cells (e.g., neural) has been established. Additionally, each worm is an exact copy of the parent worm, without variation, so its development is unwaveringly predictable. But perhaps the most remarkable thing is that, despite its minimalist structure, C. elegans exhibits behavior. If you touch it, it crawls away, but it also crawls toward, and discriminates among, different odors.

Shroff says, “It’s remarkable that something this simple exhibits specific behavior, and even though we know a lot about this little guy, there is so much we don’t know. My goal is to make a movie of actual brain formation in one of the simplest of organisms we can study.”

Again, one might ask what this has to do with human health. Shroff explains, “The question is, how does a brain get wired? Some people study mouse brains, and others study human brains, and there are great arguments for doing all that. But I would argue that we should also try to get a solid understanding of the simplest brain—completely, or as much as we can. So much is already understood about C. elegans. You can put it under a microscope and you can see it develop, but we want to figure out how this happens—from the point when it is just two cells, to the point that it looks like a worm. I believe there is a very fundamental, yet tangible element between this and the development of the human brain, and C. elegans presents a good ‘back to the basics’ starting point to discover how the brain forms.”

A mental comparison of two very different biological “haystacks” is called to mind as Shroff says, “C. elegans has only 302 neurons. A human brain has something like 10 billion neurons, each of which may have somewhere around 7,000 synapses. I’m interested in the larval worm, how each of its neurons gets to where it needs to go, and how those neurons talk to other neurons. These studies just might reveal solid clues about how neurons find their respective paths to where they need to be in human brains, as well as other higher organisms.”

Chicken or Egg? Microscope or Mutant?

As he settles in at his computer to review some research images, Dr. George Patterson, the other partner in this Intramural duo, proudly mentions he has, just the night before, learned some dance moves from Slumdog Millionaire. Quickly back to task, he begins to describe the dynamic of the two labs, and it becomes obvious that, by inherent design, success in one lab feeds the success of the other.

“We have a practical, valuable alliance. It’s kind of like a power drill and drill bits. Hari develops the instrumentation, and I develop other pieces of the microscopy imaging puzzle that are essential for optimizing things he’s developing. Furthermore, because we’re coming from different perspectives with the same goals, we can push this technique forward very efficiently relying on each other’s in-depth expertise.”

Patterson’s role is reminiscent of the James Bond character Q (i.e., Quartermaster) as he invents and supplies very specialized tools that allow investigators to see the precise location, movement, and interactions of specific molecules within cells. With the echo of Shroff’s passion and sheer fascination for nano exploration plainly audible in his dialogue, Patterson says, “The great thing about these fluorescent proteins is that they’re genetically encoded. That is, we attach one piece of DNA to the piece of DNA that is the protein we want to study. This not only gives us an up-close look at cell structure, but it also lets us see what’s going on within living cells.”

As he continues, one realizes there’s a lot more to think about than just making pretty, glowing molecules. “In this case, we used a temperature-sensitive molecule. At a little higher temperature, proteins fold in a way that makes them get stuck in a cellular subsystem called the endoplasmic reticulum. But as we lower the temperature, the proteins fold properly and move on to the Golgi apparatus, then to other parts of the cell. So this tool helps us see and study the cellular secretion pathway and process.”

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Designer Molecules

Patterson’s work essentially boils down to crafting variations on the general fluorescent protein theme, giving them chemical properties tailored to researchers’ specific interests. As for Patterson’s own GFP, the first photoactivatable protein, he says, “Dark to light contrast in an image helps us see. It’s like when you use a telescope, you want to get far away where the sky is black and there is little or no competition from city lights, and there, you can see the dimmest stars. On that same kind of principle, we modified my GFP to dim prior to activation, and the darker background helps us locate and monitor the actual items of interest in a cell.”

Pointing to a new image on his computer, “This shows an organelle called a lysosome, which is like a cellular garbage disposal that chews up proteins. The dim, dark background allows you to pinpoint the fluorescence better and track location and spacing of the lysosomes, as well as get a sense of the structural development and changes.”

Similar glowing proteins now come in a range of fashion colors—from near infrared, to violet. “You have to be creative. Depending on the question we’re trying to answer, we might need a new molecule that will do something different than the one we have, so we design a molecule to do just exactly what we want. And if we want to study dynamic interactions of two things within a cell, we might use two different colored proteins.”

Simiply Brilliant!

Mentioning research done about a decade ago at the California Institute of Technology, France, and elsewhere, that resulted in the creation of fluorescing animals, such as mice, rabbits, and zebra fish, Patterson reports that his current work, although fledgling on the research horizon, involves investigation of HIV, polio, and phosphate-related disease states, among others. “We have a very exciting collaboration with NCI in Frederick that has implications for HIV. We are looking at the behavior of molecules—where are they located at various stages of infection, which molecules get recruited at each step, and how organelles are affected. We are discovering where it’s replicating inside the cell, and how the virus hijacks a lot of the cell’s machinery.”

Another collaboration with Clara Waterman-Storr of NHLBI investigates cell motility. “Except for the blood, people don’t really think about their cells being mobile, but a lot of cells are moving around your body at any given moment.” Patterson fires up another image and explains that the focal adhesion is a kind of foot the cell uses to move by forming sticky adhesion points toward which the rest of the cytoskeleton subsequently flows. Job security seems to be a non-issue in Patterson’s field as he continues his slide show, describing the seeming endless turns his research has, and might take.

Greeks and Geeks

The research trail can be dusty and tiresome, but at the end of the day, Patterson wouldn’t wish to be anywhere else. He says, “We have evaluated all the available molecules, and it can be really frustrating. Some work well, in general; some work well only in certain temperatures or pH environments; and some work better than others on the same application. But you have to approach it knowing that none of it is a waste of time. Even when things don’t work the way you anticipated, that mere exercise, whatever you did, taught you something new. And further down the line, you might be working on something else that makes that proverbial light bulb come on, and then you pull that ‘failed’ sample out of the refrigerator, and wind up successful with something entirely different.”

Such is the nature of the research beast, whose greatest challenges foster the deepest appreciation when ‘eureka’ moments of discovery come wrapped in unexpected epiphany.
 

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