Researchers have developed a brand-new microscopy strategy called BonFIRE, which integrates fluorescence microscopy and vibrational microscopy to envision biological processes at the single-molecule level. This ingenious technique permits greater selectivity and level of sensitivity, enabling scientists to image particles with vibrational contrast. Credit: Caltech
Caltech scientists have developed BonFIRE, a cutting-edge microscopy technique that combines fluorescence and vibrational microscopy. This brand-new technique offers exceptional single-molecule imaging and uses isotopes to create different vibrational colors, supplying deep insights into biological molecules and procedures.
If you picture yourself peering through a microscopic lense, you probably image taking a look at a glass slide with an amoeba, or possibly a human cell, or possibly even a little bug of some kind.
However microscopic lens can see much more than these small living things, and a new type of microscopy developed at the California Institute of Technology (Caltech) is making it simpler to see the really particles that comprise living things.
Researchers have developed a new microscopy method called BonFIRE, which combines fluorescence microscopy and vibrational microscopy to envision biological procedures at the single-molecule level. The other method is vibrational microscopy, which makes use of natural vibrations in the bonds that hold together the atoms of a molecule. That bombardment causes the bonds of the products molecules to vibrate in such a way that their type can be recognized. The procedure works like this: The sample is very first stained with a fluorescent color that bonds to the particles planned to be imaged.” Unlike conventional fluorescence microscopy, which can just identify a handful of colors at a time, BonFIRE utilizes infrared light to thrill different chemical bonds and produces a rainbow of vibrational colors,” Wei says.
The BonFIRE Technique
In a paper published in the journal Nature Photonics, researchers from the laboratory of Lu Wei, assistant professor of chemistry and private investigator with the Heritage Medical Research Institute, demonstrate what they are calling bond-selective fluorescence-detected infrared-excited spectro-microscopy, or BonFIRE.
BonFIRE integrates two microscopy strategies into one procedure with greater selectivity and sensitivity, enabling scientists to picture biological procedures at the unprecedented single-molecule level and understand biological systems from a molecular point of view.
” With our brand-new microscope, we can now picture single molecules with vibrational contrast, which is challenging to do with existing technologies,” states Dongkwan Lee, study co-author and chemical engineering college student.
Postodoctoral scholar Haomin Wang (left) and college student Dongkwan Lee (best) demonstrate operation of the BonFIRE microscopy apparatus. Credit: Caltech
Methods Behind BonFIRE
One technique associated with BonFIRE is fluorescence microscopy, which images particles and other microscopic structures by tagging them with fluorescent chemical markers, causing them to glow when imaged.
The other strategy is vibrational microscopy, which makes use of natural vibrations in the bonds that hold together the atoms of a particle. That barrage causes the bonds of the products particles to vibrate in such a method that their type can be determined.
Lu Wei, assistant teacher of chemistry and private investigator with the Heritage Medical Research Institute. Credit: Caltech
Integrating Strengths
Wei says that fluorescence microscopy enables scientists to observe single particles, however it does not supply abundant chemical details. On the other hand, vibrational microscopy provides that rich chemical info however only works when the molecule being imaged exists in big amounts.
BonFIRE navigates these restrictions by coupling vibrations to fluorescence, efficiently integrating the strengths of the two techniques. The process works like this: The sample is first stained with a fluorescent color that bonds to the molecules intended to be imaged. The sample is then bombarded by a pulse of infrared light, whose frequency is tuned to delight a specific bond discovered because color. As soon as the bond is excited by just a single photon of that light, a second higher-energy pulse of light shines on it and delights it to fluoresce with a radiance that can be found by the microscopic lense. In this way, the microscope can image entire cells or single particles.
Future Prospects
” We are interested by this spectroscopy procedure and are delighted to turn it into a novel tool for contemporary bioimaging,” states Haomin Wang, study co-author and postdoctoral scholar research associate in chemistry. “Over the past three years, we have actually been on an adventure to build our custom-made BonFIRE microscopic lense and gain deeper understanding on this spectroscopic procedure, which further helped us to enhance each element in our setup to reach the performance we have now.”
In their paper, the scientists likewise show the ability to tag biomolecules with “colors,” permitting them to be separated from each other. This is done by using numerous isotopes of the atoms that make up the dye particle. Since their nuclei have greater or fewer neutrons), (Isotopes are forms of an element with various atomic weights. The frequency at which their bonds vibrate modifications with the increased or decreased mass of the atoms.
” Unlike conventional fluorescence microscopy, which can just differentiate a handful of colors at a time, BonFIRE uses infrared light to delight various chemical bonds and produces a rainbow of vibrational colors,” Wei states. “You can identify and image numerous various targets from the exact same sample at a time and expose the molecular diversity of life in spectacular information. We hope to be able to demonstrate the imaging capability with tens of colors in live cells in the future.”
Reference: “Bond Selective Fluorescence Imaging with Single Molecule Sensitivity” by Haomin Wang, Dongkwan Lee, Yulu Cao, Xiaotian Bi, Jiajun Du, Kun Miao and Lu Wei, 29 June 2023, Nature Photonics.DOI: 10.1038/ s41566-023-01243-8.
Additional co-authors are chemistry college students Yulu Cao, Xiaotian Bi, Jiajun Du, and Kun Miao.
Funding for the research study was offered by the National Institutes of Health and the Alfred P. Sloan Foundation.