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Microscopy Method Uses Infrared Light to Image Molecules with Vibrational Contrast

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The complementary strengths of fluorescence and vibrational microscopy are combined in a new technique developed at Caltech, which is called bond-selective fluorescence-detected infrared-excited spectro-microscopy (BonFIRE). BonFIRE will benefit biological investigations by providing researchers with rich chemical information as well as single-molecule sensitivity.

“With our new microscope, we can now visualize single molecules with vibrational contrast, which is challenging to do with existing technologies,” researcher Dongkwan Lee said.

Fluorescence microscopy allows researchers to observe single molecules, but it does not provide detailed chemical information about molecular distributions. Vibrational microscopy provides rich chemical specificity, but only works when the molecule being imaged is present in large amounts.
Postodoctoral scholar Haomin Wang (left) and graduate student Dongkwan Lee (right) demonstrate the operation of the BonFIRE microscopy apparatus. Courtesy of Caltech.
Postdoctoral scholar Haomin Wang (left) and graduate student Dongkwan Lee demonstrate the operation of the BonFIRE microscopy apparatus. Courtesy of Caltech.

BonFIRE links vibrations to fluorescence. It uses two-photon excitation in the mid- and near-infrared to upconvert vibrational excitations to electronic states for fluorescence detection, thus encoding vibrational information into fluorescence.

First, the sample is stained with a fluorescent dye that bonds to the molecules to be imaged. The sample is then irradiated with a pulse of infrared light. The frequency of the light is tuned to excite a specific bond in the dye. Once the bond is excited by a single photon of the IR light, it is irradiated with a second, higher-energy pulse, which excites the bond to fluoresce with a glow that can be detected by the BonFIRE microscope. This approach enables the researchers to use BonFIRE to image entire cells or single molecules.

The tunable, narrowband picosecond pulses used by BonFIRE ensure high sensitivity, biocompatibility, and robustness for bond-selective biological interrogations over a wide range of reporter molecules.

The researchers demonstrated BonFIRE spectral imaging in both fingerprint and cell-silent spectroscopic windows with single-molecule sensitivity for common fluorescent dyes. They demonstrated BonFIRE’s imaging capabilities on various intracellular targets in fixed and live cells (neurons and tissues), with the promise for further vibrational multiplexing. To show dynamic bioanalysis in living systems, the researchers implemented a high-frequency modulation scheme and demonstrated time-lapse BonFIRE microscopy of live HeLa cells.

The ability to tag biomolecules with colored dyes, in order to differentiate one molecule from another, is done by using isotopes of the atoms that make up the dye molecule. The frequency at which the bonds of the dye molecule vibrate will change as the mass of atoms is increased or decreased.

“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,” professor Lu Wei said. “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.”
Lu Wei, assistant professor of chemistry at Caltech and investigator with the Heritage Medical Research Institute. Courtesy of Caltech.
Lu Wei, assistant professor of chemistry at Caltech and investigator with the Heritage Medical Research Institute. Courtesy of Caltech.

In the future, Wei and her team hope to demonstrate the imaging capability of BonFIRE with tens of colors in live cells.

“We are fascinated by this spectroscopy process and are excited to turn it into a novel tool for modern bioimaging,” researcher Haomin Wang said. “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.”

With BonFIRE, the Caltech team expands the bioimaging toolbox by providing a new level of bond-specific vibrational information and greater sensitivity and selectivity at the molecular level than either fluorescence or vibrational microscopy can alone provide.

The research was published in Nature Photonics (

Sep/Oct 2023
infrared spectroscopy
The measurement of the ability of matter to absorb, transmit or reflect infrared radiation and the relating of the resultant data to chemical structure.
fluorescence microscopy
Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction.
Research & TechnologyeducationAmericasCaltechCalifornia Institute of TechnologyimagingSensors & Detectorsspectroscopyinfrared spectroscopyopticslight sourcesMicroscopyfluorescence microscopyvibrational microscopyBiophotonicsmedicalpharmaceuticalmolecular microscopyBioScan

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