NIST modifies IR microscopy for improved live cell imaging

11 Sep 2024

Quantum cascade laser and absorption compensation method captures clearer views of biomolecules.

Imaging live cells without disrupting their natural behavior or damaging their structures is one key challenge in biomedical optics, and has been tackled in multiple ways.

A project at NIST has now developed a new infra-red imaging technique intended to allow capture clear images of biomolecules inside cells, and has published the findings in Analytical Chemistry.

The goal was to overcome the tendency of water within cells to absorb infra-red radiation, a hurdle to obtaining precise imaging data about the proteins in the cell.

NIST's new method removes the obscuring effects of water in IR-based measurements and allows researchers to determine the amounts of key biomolecules in cells, such as the proteins that direct cell function.

An ability to measure the changes in these amounts inside living cells could speed up advances in biomanufacturing, cell therapy development and drug development, according to the researchers.

"In the spectrum, water absorbs infrared so strongly, and we want to see the absorption spectrum of proteins through the thick water background," commented Young Jong Lee of NIST. "So we designed the optical system to uncloak the water contribution and reveal the protein signals."

Drug screening and cancer therapy

The approach taken by the NIST Biomaterials Group included the development of solvent absorption compensation (SAC), implemented in a benchtop IR microscope based on an external-cavity quantum cascade laser.

SAC-IR microscopy adjusts the incident light using a pair of polarizers to precompensate the IR absorption by water while retaining the full dynamic range, according to the team's published paper. Integrating the IR absorbance over a cell can also yield the total mass of biomolecules per cell.

NIST constructed its platform around a hand-built IR laser microscope and applied it to the imaging of the fibroblast cells making up connective tissue in living samples. Over a 12-hour observation period, researchers were able to identify proteins, lipids and nucleic acids during stages of the cell cycle such as cell division, doing so faster than current alternative techniques according to the project.

The SAC-IR method also successfully measured the absolute mass of proteins in a cell, in addition to nucleic acids, lipids and carbohydrates. This capability could help establish a foundation for standardizing methods of measuring biomolecules in cells, potentially valuable in biology, medicine and biotechnology.

Other applications could include during use of cells for drug screening, either in discovery of new drugs or in understanding the safety and efficacy of a drug candidate. SAC-IR might help to assess the potency of new drugs by measuring absolute concentrations of various biomolecules in a large number of individual cells, or to analyze how different types of cells react to the drugs.

"In cancer cell therapy, for example, when cells from a patient’s immune system are modified to better recognize and kill cancer cells before being reintroduced back to the patient, one must ask, 'Are these cells safe and effective?'," commented Young Jong Lee.

"Our method can be helpful by providing additional insight with respect to biomolecular changes in the cells to assess cell health."