Light it Up

Modern-day physicians have lots of ways to look inside the human body. X-ray machines show us broken bones, ultrasound scans monitor unborn babies in the womb, and CT (computed tomography) and MRI (magnetic resonance imaging) scans can reveal damaged soft tissues, from ruptured appendices to brain tumors.

As beneficial as many of these diagnostic tools are, they do come with some consequences.

The high-energy photons used in some of these tests can damage healthy cells. For this reason, many chemists around the world are working hard to advance a different approach to bioimaging: one based on visible, less damaging wavelengths of light. To do this, chemists are designing and synthesizing many different light emitting—fluorescent—molecules and  materials. 

“With fluorescence you can directly see what’s going on, especially under a microscope,” says Xiaogang Liu, a chemist at the Singapore University of Technology and Design. A particular advantage of fluorescence bioimaging is that our eyes are very sensitive to visible light, Liu says.

Whether it’s our computer screen, our television, or a movie, our eyes can easily distinguish between standard- and high-definition resolution. The high- definition resolution of fluorescence bioimaging allows scientists to view really small cells, or even tiny features of cells, under a microscope.

Using fluorescent dyes, we can track cells and fluids as they move or change, “and then we can record a movie,” points out Eva Hemmer, a chemist at the University of Ottawa. “We can’t do that with an MRI, a CT, or an X-ray scan.”

Making Molecules Shine

Fluorescent materials are ubiquitous, such as the familiar long tubes of fluorescent light bulbs or quantum dots. Another Nobel-winning fluorescent  material is the green fluorescent protein (GFP) first isolated from jellyfish in the 1960s. 

But these two classes of fluorescent materials are limited when it comes to imaging human tissue or the human body. “We cannot inject quantum dots into our bodies, and we cannot modify our DNA to produce GFP,” says Liu. So, chemists have been hard at work designing fluorescent molecules and nanoparticles that are more biocompatible and easier to deploy.

Thankfully, fluorescence is not a rare feature; it occurs naturally among many small organic molecules. A classic example can be found in tonic water, says Liu. Tonic water contains the molecule quinine, which glows a bright cyan (blue-green color), when exposed to ultraviolet light.

Molecules don’t glow on their own, and not every molecule produces a glow. First, to produce light, a molecule must absorb—take in—energy. Typically, fluorescent dyes absorb light of a higher energy on the electromagnetic spectrum, such as invisible ultraviolet light. Some of the energy absorbed by the excited electron is lost as the atoms shake or vibrate, and then, when the electron returns to the ground state, light of a lower energy, such as visible light, is emitted—given off.

Chemists would say that the molecules become “excited” from their ground state as they absorb the ultraviolet light, and then “relax” and fall back to their ground state glowing, or producing visible light.

Having the right structure is vital to the emission of light. After molecules are excited, they can shed excess energy by either releasing heat or light. “If a dye is very flexible, very floppy, it will have a lot of vibrations after it goes to the excited state,” explains Liu. “This kind of dye will release too much energy as heat.” 

Molecules that shed their excitation energy as light rather than heat tend to be rigid or confined, which means they cannot easily relax by vibrating and releasing heat. Instead, they are forced to relax by emitting light and they glow.

Shining as a Crowd

A leading design of fluorescent dyes originated from the laboratory of Ben Zhong Tang at the Hong Kong University of Science and Technology decades ago. These molecules are shaped like rotors or windmills, carrying “blades” or “paddles” that have some flexibility to rotate. By accident, Tang and his students discovered that these molecules would shine brightly when lots of them were concentrated together. In close quarters, the molecules’ motions became restricted, which in turn forced them to shed their energy as light and not as heat or motion. The researchers dubbed this phenomenon aggregation-induced emission (AIE), and the molecules were given a name: AIE-gens.

But by themselves, AIE-gens are not very useful in biomedical settings. “That’s because they are hydrophobic, and therefore they don’t easily dissolve in water,” says Jax Lee, a cofounder of NanoLumi, a Singapore-based start-up company making fluorescent materials.

To turn a molecule into a useful product for bioimaging, NanoLumi researchers pack AIE-gens into biocompatible nano-sized shells. These shells are hydrophobic (water-hating) on the inside to hold AIE-gens, and hydrophilic (water-loving) on the outside, so they can enter biological cells. The result is ultra-bright nanoparticles, which the researchers call AIE-dots.

Packing these dyes into nanoshells helps stabilize them so they glow for longer periods of time. Also, the shells can be decorated with groups that send them to specific sites within the body. Fluorescent dyes should stain only specific targets of interest, such as certain proteins, mem-branes, or tumors. “We don’t want the dye to go everywhere, only the target that we are interested in,” says Liu. 

Beyond Skin Deep

To image directly through skin, however, scientists can’t really rely on fluorescent dyes that are excited by ultraviolet light and emit visible light. Neither type of light can pass through skin, and ultraviolet light is harmful to our skin.

“Humans are mainly water, tissue, skin, fat, and all kinds of muscles,” says Hemmer. “All this stuff can absorb light, so chemists need to create dyes that operate in a more suitable range of wavelengths.”

That range turns out to be in the near-in-frared region, that is, light that has about 1,000 nanometer (nm) to about 2,000 nm wavelengths, which is very close to the visible region but just outside what we can see, according to Hemmer. 

But near-infrared light contains less energy than UV or visible light—remember, wavelength and energy are inversely proportional (E=hc/λ where E is energy, h is Planck’s constant, c is the speed of light, and λ is the wavelength): longer wavelengths of light mean lower energy. The challenge now is to find elements that would fluoresce when exposed to this range of wavelengths. To create the desired visible glow using lower-energy light from the infrared region, chemists turned to a group of elements near the bottom of the Periodic Table, the lanthanides.

“Lanthanide elements are very special,” Hemmer says. While other fluorescent materials are generally excited just once before they relax, the lanthanides, because of their unusual electronic structure, can be excited in a step-by-step manner. 

Hemmer compares this excitation process to a person taking a small step up a ladder, then waiting for a tiny fraction of a second, before they take another step. In other words, lanthanides can be excited twice, or even more times and their excitation energy adds up. When released, the energy emitted is often at higher energy, shorter wavelengths.

This process is known as upconversion, because the energy that is absorbed starts out at longer wavelengths (near-infrared 1,000 nm to 2,500 nm) and lower energy, but is then emitted at higher energy, shorter wavelength (visible 400 nm to 700 nm). The energy of the light is upconverted to higher energy rather than downconverted to lower energy in more traditional absorption-emission systems.

Chemists are already thinking about designing multi-purpose nanoparticles to respond differently to different triggers. “It’s the same nanoparticle, but it could be used for diagnostics or a therapeutic agent,” Hemmer says. There’s still a lot of research to be done, however.

New devices would need to be engineered, and researchers are still exploring which wavelengths of light would be most effective for bioimaging versus for therapy, and how to strike the right balance. Physicists would first study how light interacts with biological tissues, then chemists could design fluorescent dyes that address the requirements that the physicists have identified. “This is where physicists and chemists need to work really closely together,” says Hemmer.