Professor Karel Svoboda won the Brain Prize in 2015, together with Winfried Denk, Arthur Konnerth, and David Tank “for invention, refinement and use of two-photon microscopy to provide detailed, dynamic images of activity in individual nerve cells, dendrites and synapses, thereby transforming the study of development, plasticity and functional circuitry of the brain”.

The Brain Prize is an international award that recognizes and celebrates highly original and groundbreaking advances in any area of brain research, from basic neuroscience to applied clinical research. Since it was founded in 2011 and up until 2025, The Brain Prize has been awarded to 49 scientists from 11 countries.

Why I Study the Brain

Science is driven by a deep curiosity to explore nature and the universe. One of the most fundamental questions scientists ask is: how does the brain create our experiences and behaviors, in the form of thoughts, feelings, perceptions, and actions? We still know very little about the brain—we do not fully understand how its parts work on their own, or how they work together to process information and produce behavior. This is especially true in brain disorders, where science has made only limited progress in understanding their causes and therefore has produced few cures. Take Parkinson’s disease, for example—a movement disorder that often affects elderly people. Doctors have used a medicine called levodopa to treat its symptoms for about 50 years, yet we do not actually understand why it works!

While there is a great need to understand the brain better, especially to help people with brain disorders, it is also important and exciting to study the normally functioning brain. I am strongly drawn to brain science because it feels like a great adventure into one of the biggest remaining mysteries Each time brain scientists develop a new tool, we learn something fundamentally new about the brain. That is why I am interested in tool development, and why I believe that new tools have driven brain science forward—even more so than in other areas of biology.

Seeing Inside The Brain With Two-Photon Microscopy

One way to study how the brain works is to watch it working in real time. But looking inside living tissue, like an animal’s brain, is very challenging. Tissue makes it hard for light to travel deep inside—and also hard for light to come back out. If you look at your hand, you cannot see inside it, right? That is because the skin blocks most light from passing through. The same happens in brain tissue. This is a big problem for microscopes, because microscopes need light to “see”. Traditional microscopes work well for a single cell in a dish or for a very thin slice of tissue because light can easily pass through. But most of biology—and much of the most interesting brain activity—happens in thick, living tissues, which require a different approach.

This is where two-photon microscopy comes in [1]. Photons are particles of light—in this case, tiny packets of energy emitted by a laser. When a photon with the right energy hits a molecule, it can be absorbed and cause an excitation, meaning the molecule briefly jumps to a higher energy state. In two-photon microscopy, something special happens: two photons, each with half the required energy, arrive at almost the same time and combine their energy to excite a fluorescent molecule that glows after it absorbs light. This two-photon effect happens only where the light is extremely concentrated—at the tiny point where the laser is focused. Because excitation happens only at the focal point, we get a signal (meaning we see the fluorescent molecule “glowing”) mainly from that tiny spot—not from all the tissue the light passes through on its way there.

Two-photon microscopy has important advantages over traditional microscopes (Figure 1). First, it uses lower-energy light, which is gentler and less damaging to living tissues, so scientists can image for longer periods of time [2]. Second, this light has a longer wavelength, which helps it travel deeper into tissue—often up to about 1 mm, which is roughly five times deeper than a traditional light microscope. Third, because fluorescence is produced mainly at the focal spot, the images are sharper and more precise, helping scientists see fine details inside living brain tissue.

Developing Two-Photon Microscopy For Brain Research

The original idea for two-photon microscopy was not developed specifically to look inside brain tissue. In the late 1980s at Cornell University (New York, USA), one group had a laser-scanning microscope, and another group had the right kind of laser for two-photon microscopy. They published the first two-photon images in 1989 (Figure 2A) [3]. At the time, they did not know what two-photon microscopy would be most useful for—so the paper did not highlight the uses in intact tissue that eventually made the method famous. Later, when I was at Bell Laboratories working with Winfried Denk, David Tank and other colleagues, we realized what two-photon microscopy was truly great for imaging deep inside the living brain (Figure 2B).

Once we recognized that potential, we faced a new set of engineering challenges. We had to rethink microscope design from the ground up. Traditional microscopes were built for cells in dishes, not for studying whole, live animals. So, we built microscopes on strong lab tables, and sometimes even hung the microscope above the animal so it could look down while the animal moved. We also developed software to control the microscope and synchronize it with other measurements—like video cameras that track what the animal is doing.

In the early days, we even built our own lasers from kits. A typical experiment often meant spending most of the time just keeping the laser running—and then, once it finally worked, doing the experiment quickly before something broke again. Thankfully, that changed as two-photon microscopy became more common and laser manufacturers began selling dependable systems (Figure 2C).

In a typical setup, the laser stays on a single tiny spot for about 100 ns (a nanosecond is one billionth of a second), delivering up to 10¹⁰ (10 billion, which is ten thousand million) photons to that spot to create the measurable signal there. To scan across the tissue, tiny mirrors tilt back and forth extremely quickly—up to 20,000 times per second—steering the laser spot across the sample so the microscope can build up an image line by line. It is an impressive piece of engineering (Figure 3).

In addition to the laser and microscope hardware, we had to solve another crucial problem: creating the right fluorescent molecules for brain cells. At the time, existing fluorescent molecules did not work well for measuring activity in the living brain. I devoted about 10 years to developing molecules that clearly report brain-cell activity and can be introduced into brain cells without harming them [4]. Once these technical pieces came together—lasers, scanning microscopes, software, and fluorescent molecules—modern two-photon microscopy became a reality, and it is now one of the most widely used tools in basic brain research.

Seeing the Small and the Large at the Same Time

One of the things that interests me most is understanding how learning happens in the brain. Learning involves changes in the connections between brain cells, and it often involves large areas across the brain. This creates a major challenge: how can we see the smallest parts of brain cells—their connections—while also watching many brain cells at once? Most microscopes face a trade-off between resolution (seeing tiny details) and field of view (seeing a large area). If you zoom in for high resolution, you can only view a small patch. If you zoom out to see a large area, you lose the fine details.

Using modern design methods, including machine learning, we developed special lenses that can capture both the small and the large at the same time. We call this high etendue, which means the system can collect a lot of light from a large area at once—so we can image large scenes of brain activity without giving up too much detail [5]. One lens we recently developed can view an area 5 mm across—about the size of an entire mouse brain—while still achieving a very high resolution. This lets us see tiny structures like individual brain-cell connections across many brain regions at once—more than 10 times the information we could get using typical microscope lenses. This is exciting because it brings us closer to watching learning unfold in real time, and to discovering how whole networks of brain cells reorganize themselves to produce new behavior.

The Big Picture: Curiosity-Driven Research Changes the World

Many of the biggest breakthroughs in science and technology begin with curiosity, not with a plan to solve a practical problem. Two-photon microscopy is a great example. It grew out of physicists developing new kinds of lasers simply to explore what they could do—long before anyone realized such lasers could become a powerful way to look deep inside the brain. A similar story happened with glowing proteins: the green fluorescent protein came from research on how jellyfish glow, and it eventually led to fluorescent molecules, which are now used throughout biology, including in two-photon microscopy. Early artificial intelligence also grew from researchers trying to understand how learning might work in brains, and those ideas eventually led to today’s powerful AI tools like ChatGPT.

The pattern is clear: when clever people explore interesting topics, many times real-world applications grow naturally from their discoveries—often in ways nobody could have predicted. I encourage you to join science and explore some of the biggest mysteries that might become the basis of future technological breakthroughs we cannot even imagine yet.

Glossary

Parkinson’s Disease: ↑ A common brain disorder in older adults that makes it difficult to control movement, so people may shake, move slowly, or feel stiff.

Two-photon Microscopy: ↑ A way to see inside living tissue using a focused laser that makes cells glow only at a tiny, chosen spot, creating a sharp image without damaging surrounding tissue.

Excitation: ↑ When a molecule absorbs light and gets an “energy boost”, putting it in a higher-energy state.

Fluorescent Molecule: ↑ A tiny chemical that absorbs light and then gives off a different color of light, making it glow so scientists can see it.

Wavelength: ↑ The length of a light wave. Longer waves with a bigger wavelength can go deeper into living tissue without harming it.

Machine Learning: ↑ When computers learn patterns from many examples so they can make decisions on their own, like improving camera lenses to take sharper pictures.

High Etendue: ↑ A microscopes ability to collect lots of light from a big area at once, so we can see a wide view without losing too much detail.

Conflict of Interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

I wish to thank Or Raphael for conducting the interview which served as the basis for this paper, and for co-authoring the paper, Iris Gat for providing the figures, and Susan Debad for copyediting the manuscript.

Further Resources

• The Brain Prize 2015 -2-photon-microscopy

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