Measuring changes in heme concentration

 Early mNIRS measurements were reported in “arbitrary units” or used a “physiological calibration” (see later method). David Delpy and colleagues, together with Hamamatsu Photonics, pioneered measurements of changes in heme concentration in “real” units, enabling subsequent quantitative measurements of variables such as blood flow and oxygen consumption (see later methods).

The Beer-Lambert Law states that changes in absorbance are directly proportional to three things: the change in chromophore concentration; an intrinsic property of the chromophore at the measuring wavelength (the extinction coefficient); and the optical pathlength. With known extinction coefficients, it is then straightforward to calculate concentration changes if you use the same or a greater number of optical wavelengths than chromophores.

For example, the Hamamatsu NIRO-500 had four measuring wavelengths and measured three unknowns — oxyhemoglobin, deoxyhemoglobin and cytochrome oxidase — in units of µM·cm. To convert these to concentration changes in µM, you need to divide by the optical pathlength.

Determining that optical pathlength is the difficult part. Because multiply scattered light travels further than unscattered light, a differential pathlength factor (DPF) is needed to multiply the source–detector separation to obtain the optical pathlength. Phase-resolved spectroscopy showed that the DPF varied substantially between different tissues, with values typically between about 4 and 6.

Duncan A, et al. “Optical pathlength measurements on adult head, calf and forearm and the head of the newborn infant using phase resolved optical spectroscopy.” Physics in Medicine & Biology 40 (1995): 295–304.

For complicated reasons, the DPF is also wavelength dependent. This means that the in vivo heme spectral fit has to be “modified” from that obtained in vitro.

Essenpreis M, et al. “Spectral dependence of temporal point spread functions in human tissues.” Applied Optics 32 (1993): 418–425.

Note that some manufacturers now focus solely on tissue oxygen saturation measurements (see later methods).