If you were to use infrared (IR) transmission microscopy, you would appreciate its chemical quantification abilities—unless your object of study was a hydrated cell. In that case, you might complain about being reduced to looking through a cell, darkly. You would be frustrated that water absorbs IR light so well that anything in the cell other than water is practically invisible.
To reveal biomolecular reality more completely, scientists at the National Institute of Standards and Technology (NIST) led by research chemist Young Jong Lee, PhD, have developed a new method called solvent absorption compensation (SAC) IR microscopy. The method, which employs an external-cavity quantum cascade laser, adjusts the incident light using a pair of polarizers to precompensate the IR absorption by water while retaining the full dynamic range.
Essentially, SAC IR microscopy 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.
With a hand-built IR laser microscope, the NIST scientists showed how SAC IR microscopy can be used to image fibroblasts, cells that support the formation of connective tissue. The apparatus worked so well that the scientists now anticipate that SAC IR microscopy could be used to accelerate advances in biomanufacturing, cell therapy development, drug development, and more.
Details about the application appeared in Analytical Chemistry, in an article titled, “Benchtop IR Imaging of Live Cells: Monitoring the Total Mass of Biomolecules in Single Cells.”
“Integrating the IR absorbance over a cell yields the total mass of biomolecules per cell,” the scientists reported. “We monitor the total mass of the biomolecules of live fibroblast cells over 12 h, demonstrating promise for advancing our understanding of the biomolecular processes occurring in live cells on the single-cell level.”
“In the spectrum, water absorbs infrared so strongly, and we want to see the absorption spectrum of proteins through the thick water background,” Lee said. “So, we designed the optical system to uncloak the water contribution and reveal the protein signals.”
He added that SAC IR microscopy could help establish a foundation for standardizing methods for measuring biomolecules in cells, which could prove useful in biology, medicine, and biotechnology: “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?’ Our method can be helpful by providing additional insight with respect to biomolecular changes in the cells to assess cell health.”
Other potential applications include using cells for drug screening, either in discovery of new drugs or in understanding the safety and efficacy of a drug candidate. For example, this method could 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.
The researchers hope to develop the technique further so it can measure other key biomolecules, such as DNA and RNA, with greater accuracy. The technique could also help provide detailed answers to fundamental questions in cell biology, such as what biomolecule signatures correspond with cell viability.
“Some cells are preserved in a frozen state for months or years, then thawed for later use,” Lee noted. “We don’t yet fully understand how best to thaw the cells while maintaining maximum viability. With our new measurement capabilities, we may be able to develop better processes for cell freezing and thawing by looking at their infrared spectra.”
