How fNIRS Extracts a Signal

In the previous Lesson, you learned that the Optical Window exists because the main tissue chromophores have their lowest combined absorption between 650 and 950 nm. You also learned that hemoglobin comes in two forms, oxygenated and deoxygenated, and that these two forms absorb light differently. But we left that idea unexplored, so we will unpack it.

1. What is (Light) Spectroscopy?

Every material interacts with light in its own characteristic way. Some materials absorb certain wavelengths strongly and let others pass through. Some reflect, some transmit, some scatter. This selective interaction with light is not random; it is determined by the molecular structure of the material, and it is reproducible and quantifiable. Light Spectroscopy is the science of measuring these interactions: shining light at a sample and analyzing which wavelengths are absorbed, reflected, or transmitted to reveal what the sample is made of and in what quantity. (Note, by this definition, you probably already guessed that there are other types of spectroscopy that explore other properties of materials, such as mechanical, electric, and magnetic properties.)

You already encounter spectroscopy in everyday life, even if you don’t call it that. Leaves look green because the chlorophyll in them strongly absorbs red and blue light, reflecting green light. Your blood looks bright red when oxygenated and darker red when deoxygenated, because the two forms of hemoglobin absorb visible light differently. The light your eyes receive carries chemical information, and spectroscopy is the formalized, quantitative version of reading that information.

In fNIRS, the sample is tissue, the light is near-infrared, and the molecules we are trying to read are the hemoglobin species inside blood vessels beneath the scalp.

2. Hemoglobin: The Molecule at the Center of fNIRS

Hemoglobin is a protein found inside red blood cells. Its primary job is to carry oxygen from the lungs to every tissue in the body, and to return carbon dioxide back to the lungs. Each red blood cell contains hundreds of millions of hemoglobin molecules, making it essentially a specialized oxygen-transport container.

At the structural heart of each hemoglobin molecule is a component called the heme group: a ring-shaped structure containing a single iron atom. It is this iron atom that binds to oxygen. Each hemoglobin molecule has four heme groups, meaning it can carry up to four oxygen molecules at once.

Two forms, one molecule

The binding of oxygen to hemoglobin is reversible. In the lungs, where oxygen is plentiful, hemoglobin picks it up and becomes oxy-hemoglobin (HbO₂). As the blood circulates and reaches tissues that need oxygen (e.g., a working muscle, an active brain region), hemoglobin releases its oxygen and becomes deoxy-hemoglobin (HbR). The cycle then repeats as the blood returns to the lungs.

This oxygen-binding cycle causes a small but significant change in the molecular structure of hemoglobin, specifically around the heme group and the iron atom. And this structural change, crucially, alters how hemoglobin absorbs light. HbO₂ and HbR have different optical properties: they each absorb light of different wavelengths with different “efficiencies”. This difference is the physical basis of the entire fNIRS measurement.

3. Two Molecules, Two Spectra

When scientists measure how much light HbO₂ and HbR absorb across a range of wavelengths, they produce an absorption spectrum for each, i.e., a graph showing absorption on the vertical axis and wavelength on the horizontal axis. These spectra are the fingerprints of the two molecules, and they are the most important graphs in fNIRS.

In the NIR Optical Window (~650–950 nm), absorption drops significantly for both forms, but they do not drop to the same level, and they drop differently: below about 800 nm, HbR absorbs more than HbO₂. Above about 800 nm, HbO₂ absorbs more than HbR. In addition, the two curves cross at approximately 805 nm. At this specific wavelength, HbO₂ and HbR absorb light equally. This crossing point is called the isosbestic point.

These spectral differences are the key. Because HbO₂ and HbR absorb NIR light differently at different wavelengths, changes in their concentrations (caused, for example, by an increase in brain activity) produce measurable changes in the intensity of light arriving at your detector. More HbO₂ means more absorption at wavelengths where HbO₂ dominates, and therefore fewer photons reaching the detector at those wavelengths.

4. Why We Need (At Least) Two Wavelengths

Suppose you are measuring the attenuation of light through tissue at a single wavelength, and you observe that the signal changes. How do you know whether that change was caused by an increase in HbO₂, a decrease in HbR, or some combination of the two? At a single wavelength, you cannot tell. Both chromophores are absorbing simultaneously, and a single measurement gives you only their combined effect.

This is a problem of unknowns. You have two quantities you want to determine (the change in HbO₂ concentration and the change in HbR concentration) and one measurement is not enough to separate them. You need at least as many measurements as you have unknowns. Two wavelengths give you two measurements: two equations, two unknowns (thus, a solvable system).

The isosbestic point deserves a moment of attention, because it illuminates the logic clearly. At ~805 nm, HbO₂ and HbR absorb very close to each other. If the oxygenation state of hemoglobin changes (e.g., some HbO₂ becoming HbR or vice versa), the total hemoglobin concentration stays the same, and the absorption at 805 nm does not change at all. A measurement at this wavelength is blind to oxygenation changes. Conversely, at wavelengths far from the isosbestic point, the two forms differ most in their absorption, making those wavelengths the most informative for detecting oxygenation changes. This is why fNIRS wavelengths are chosen on opposite sides of the isosbestic point.

5. Beyond Hemoglobin

Hemoglobin is the dominant NIR chromophore in tissue, and it is the one that the vast majority of fNIRS research focuses on. But it is not the only one that matters.

If you read the Jöbsis (1977) paper mentioned in the previous Lesson, you will notice that his primary target was not hemoglobin at all; it was cytochrome c oxidase (CCO), the final enzyme in the chain of proteins inside mitochondria that produces the cell’s energy (ATP). CCO also absorbs NIR light, and its oxidation state, which changes depending on how much oxygen the cell is consuming, can in principle be tracked with the right measurement.

Why, then, is CCO not routinely measured in fNIRS studies? Two reasons. First, CCO is present in tissue at concentrations roughly three orders of magnitude lower than hemoglobin, making its contribution to the overall signal very small and easily buried in noise. Second, its absorption spectrum overlaps substantially with those of HbO₂ and HbR, making it very difficult to separate mathematically. Doing so reliably requires broadband light sources spanning many wavelengths and sophisticated analysis methods (at least well beyond what standard two-wavelength CW systems can achieve).

Measuring CCO is an active and exciting research area, because it offers a window onto cellular metabolism rather than just blood oxygenation. But it is not standard practice, and it is worth knowing it exists precisely because it reminds us that the hemoglobin-focused version of fNIRS that most researchers use is a simplification of a richer measurement problem.

Society for Functional Near-Infrared Spectroscopy.

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