New microscopy technique reveals the molecular secrets of life

When you imagine looking through a microscope, you might envision observing a glass slide with an amoeba, a human cell, or even a small insect. However, microscopes have the capability to see far beyond these tiny living entities. At Caltech, a groundbreaking type of microscopy has been developed, facilitating the visualization of the very molecules that compose living organisms.

In a recent publication in the journal Nature Photonics, scientists from Lu Wei’s laboratory, an assistant professor of chemistry and an investigator with the Heritage Medical Research Institute, unveil a novel microscopy technique known as bond-selective fluorescence-detected infrared-excited spectro-microscopy, or BonFIRE.

BonFIRE combines two microscopy methods into a single process that offers enhanced selectivity and sensitivity. This advancement enables researchers to observe biological processes at an unprecedented level—down to the individual molecule—and gain a molecular perspective on biological mechanisms.

Dongkwan Lee, a chemical engineering graduate student and co-author of the study, emphasizes the significance of the new microscope, stating, “With our new microscope, we can now visualize single molecules with vibrational contrast, which is challenging to do with existing technologies.”

Fluorescence microscopy plays a role in the BonFIRE technique. This method involves labeling molecules and other minute structures with fluorescent chemical markers, causing them to emit light when visualized.

Postodoctoral scholar Haomin Wang (left) and graduate student Dongkwan Lee (right) demonstrate operation of the BonFIRE microscopy apparatus. Credit: Caltech

Another technique employed in BonFIRE is vibrational microscopy, which leverages the inherent vibrations in the chemical bonds between atoms within a molecule. In this method, the sample under examination is exposed to infrared light, causing the bonds in its molecules to vibrate in a manner that reveals their specific types. For instance, the vibrations of a triple bond differ from those of a single bond, and the vibrations of a carbon atom bonded to another carbon atom are distinct from those of a carbon atom bonded to a nitrogen atom. This concept is akin to how a skilled guitarist can identify the plucked string and its material by listening to its tone.

Lu Wei explains that while fluorescence microscopy enables the observation of individual molecules, it lacks detailed chemical information. On the other hand, vibrational microscopy provides such chemical information but necessitates a large quantity of the molecule being imaged.

BonFIRE overcomes these limitations by linking vibrations to fluorescence, effectively combining the strengths of both techniques. Here’s how the process works: First, the sample is stained with a fluorescent dye that binds to the target molecules for imaging. Subsequently, the sample is exposed to a pulse of infrared light with a frequency tuned to excite a specific bond in the dye. When the bond is excited by a single photon of this light, a second pulse of higher-energy light illuminates it, prompting fluorescence that can be detected by the microscope. In this manner, the microscope can capture images of entire cells or individual molecules.

Haomin Wang, a co-author of the study and a postdoctoral scholar research associate in chemistry, expresses enthusiasm for this spectroscopy process and its potential as a novel bioimaging tool. Wang further explains that the researchers have spent the past three years constructing a custom BonFIRE microscope and enhancing their understanding of the spectroscopic process. These efforts have allowed them to optimize each component of their setup and achieve the performance they currently have.

The paper also demonstrates the capability of tagging biomolecules with different “colors” to differentiate them from one another. This is accomplished by utilizing various isotopes of the atoms comprising the dye molecule. Isotopes are different forms of an element with varying atomic weights due to the presence of more or fewer neutrons in their nuclei. The frequency of bond vibrations changes with the altered mass of the atoms.

Lu Wei highlights that unlike conventional fluorescence microscopy, which can only distinguish a limited number of colors simultaneously, BonFIRE employs infrared light to excite different chemical bonds, generating a range of vibrational colors. This enables the labeling and imaging of multiple targets within the same sample, unveiling the molecular diversity of life with remarkable detail. The researchers aspire to demonstrate the imaging capability with tens of colors in live cells in the near future.

Additional co-authors of the study include chemistry graduate students Yulu Cao, Xiaotian Bi, Jiajun Du, and Kun Miao.

Source: California Institute of Technology

Leave a Comment