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New Combination of Imaging Techniques Could Revolutionize Microscopy

A close-up photograph of a microscope looking at a glass slide
Credit: Logan Moreno Gutierrez / Unsplash.
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Researchers at the California Institute of Technology (Caltech) have developed a new microscopy technique that allows scientists to simultaneously observe single molecules and read their chemical information. A description and demonstration of the technique was recently published in the journal Nature Photonics

The limitations of traditional microscopy

Scientists already have access to extremely high-powered microscopes. Whether they want to study cells with a traditional optical microscope or nanoscale crystals and structures with a scanning electron microscope (SEM), there are options out there.

But while these imaging techniques give scientists a good idea of what certain objects look like, they cannot do much more than that. 

In an effort to design a new microscopy technique that puts more information into scientists’ hands, researchers at Caltech began to experiment with the possibility of combining two common laboratory techniques – vibrational spectroscopy and fluorescence microscopy – to create a new and improved microscopy method.

Vibrational spectroscopy is a popular technique in modern chemistry labs. The method hinges on the fact that chemical bonds between atoms in a molecule are not fixed – they can stretch, compress and rock back and forth. Depending on the atoms and the type of bond involved (single bond, double bond, etc.), these movements will happen at different characteristic frequencies. Crucially, when a molecule is exposed to infrared radiation, it will absorb some of the radiation at those characteristic frequencies, allowing the composition and the structure of the molecule to be pieced together. 

Fluorescence microscopy is another technique that is highly favored in modern labs, particularly in the fields of biology and biomedical science. By “tagging” substances with unique fluorescent chemical markers that glow in distinctive colors when imaged, fluorescence microscopy gives scientists access to more contrast modes than are available with traditional microscopes. 

While these two techniques are very useful in their respective fields, they also have the potential to complement each other well. Fluorescence microscopy allows scientists to observe single molecules but does not provide any additional chemical information. Vibrational microscopy has the opposite effect, affording a wealth of chemical knowledge but only when the molecules being studied are present in large amounts. 

As the Caltech researchers report in their new paper, combining these two techniques allows scientists to break down these barriers and begin looking at single molecules in more detail. 

"With our new microscope, we can now visualize single molecules with vibrational contrast, which is challenging to do with existing technologies," commented study co-author and chemical engineering graduate student, Dongkwan Lee.

Introducing the BonFIRE technique

The new technique has been named “BonFIRE” by the research team, which is short for bond-selective fluorescence-detected infrared-excited spectro-microscopy.

In the BonFIRE method, a sample is first stained with fluorescent dye in order to attach a fluorescent marker to the molecules of interest. The sample is then bombarded by a pulse of infrared light, which is tuned to a precise frequency that will excite a specific atomic bond in the fluorescent marker. Once that bond has been excited by just a single photon of infrared light, a second higher-energy pulse of radiation is shot at the sample, which causes it to fluoresce with a glow that can be detected by a microscope. In this way, the researchers say that the microscope can image entire cells or single molecules.

"We are fascinated by this spectroscopy process and are excited to turn it into a novel tool for modern bioimaging,” said Haomin Wang, study co-author and a postdoctoral scholar research associate in chemistry. "Over the past three years, we have been on an adventure to build our custom BonFIRE microscope and gain deeper understanding on this spectroscopic process, which further helped us to optimize each component in our setup to reach the performance we have now."

Colorful tags add another layer of understanding

In addition to describing their new technique, the researchers also demonstrate the flexibility of its use in the new paper. For example, they highlight how isotopes – atoms of the same element that have a different number of neutrons, and therefore a different mass – can be used to further differentiate between different target biomolecules. 

Because isotopes have different masses, the characteristic vibrations of the chemical bonds to that atom will have a slightly different frequency than normal. By attaching fluorescent dye molecules containing different isotopes to the various target molecules, it is possible for scientists to effectively “color-code” the different compositions that they are looking at.

"Unlike conventional fluorescence microscopy, which can only distinguish a handful of colors at a time, BonFIRE uses infrared light to excite different chemical bonds and produces a rainbow of vibrational colors," said principal investigator and assistant professor of chemistry, Lu Wei. "You can label and image many different targets from the same sample at a time and reveal the molecular diversity of life in stunning detail. We hope to be able to demonstrate the imaging capability with tens of colors in live cells in the near future."

Reference: Wang H, Lee D, Cao Y, et al. Bond-selective fluorescence imaging with single-molecule sensitivity. Nat Photon. Published online June 29, 2023. doi: 10.1038/s41566-023-01243-8

This article is a rework of a press release issued by the California Institute of Technology (Caltech). Material has been edited for length and content.