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A better way to do infrared microspectroscopy

Fluorescence boosts sensitivity and image quality

by Mitch Jacoby
August 5, 2021 | A version of this story appeared in Volume 99, Issue 29

A color coded micrograph of a cancer cell.
Credit: J. Am. Chem. Soc.
Various features of this cancer cell, including the plasma membrane (purple band) and regions with high protein concentration (white) stand out clearly thanks to a new imaging method.

A promising microspectroscopy technique has just become far more promising thanks to an improvement that increases the technique’s sensitivity and spatial resolution. The advance, which combines fluorescence microscopy and infrared (IR) spectroscopy, enables scientists to probe chemistry inside living cells and within tiny domains of heterogeneous materials.

Barely 5 years old, mid-IR photothermal microscopy provides molecular information from a microscopic region of a specimen by monitoring the temperature changes that result from IR-induced sample heating. The sample absorbs select bands of IR light, which triggers specific molecular vibrations that can be detected as temperature changes and used for chemical fingerprinting and sample mapping.

A color coded micrograph of a drug mixture.
Credit: J. Am. Chem. Soc.
A new imaging method reveals microscopic regions of a drug formulation enriched in ritonavir (blue) and in a polymer known as PVPVA (red).

Researchers typically probe the temperature change by monitoring how a beam of visible light, which can be focused more tightly than IR light, scatters from the sample. But scattering depends weakly on temperature, so imaging can be slow and image quality poor.

Working independently, two research teams have come up with a way to sidestep those problems. They do it by using changes in fluorescence quantum yield—a measure of fluorescence efficiency—in response to temperature change as a “thermometer” to measure IR-induced heating. The fluorescence method is about 100 times as sensitive as the scattering method, thereby enhancing data collection and image quality.

In one study, a team led by Ji-Xin Cheng of Boston University showed that by using fluorescent probes that tag specific biomolecules, the new technique can be used to pinpoint lipid droplets, phospholipid membranes, and the distribution of proteins inside a single living cancer cell (J. Am. Chem. Soc. 2021, DOI: 10.1021/jacs.1c03642). That type of information can help track cell metabolism and aid development of treatments.

In the other study, Purdue University’s Garth J. Simpson and coworkers showed that the method can be used to map—at the nanoscale—the location of pharmaceutical compounds distributed in a polymer matrix. The team applied the technique to Ritonavir, a human immunodeficiency virus drug, which is often dispersed in a water-soluble polymer to improve drug uptake. The results show microscopic phase-separated domains enriched in Ritonavir. Understanding the cause of unwanted phase separation can lead to improved formulations with longer shelf-life and higher bioavailability (J. Am. Chem. Soc. 2021, DOI: 10.1021/jacs.1c03269)

Oxana Klementieva, a biomolecular imaging specialist at Lund University, says these studies “provide a huge step forward for spectroscopy applications, especially in biomedicine.” It can be very challenging to interpret the infrared signal from tissues, cells, and other complex heterogeneous samples, she says. The fluorescently guided spectroscopy described here could help with that bottleneck because it can target specific organelles and biomolecules.

Masaru K. Kuno, a nanomaterials imaging expert at the University of Notre Dame, says it’s easy to imagine the technique being used to detect individual molecules in spectrally- and spatially-congested environments where traditional single-molecule methods fail. He points out, however, that the new technique has a key limitation. It relies on emission. Consequently, materials being investigated must have suitable emission properties and researchers must be able to measure their fluorescence quantum yields, he says..


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