Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Part 1: Prior to participant arriving to the lab

1. Ensure that the room is free of extraneous articles that may be distracting to the participant.
2. Set-up and load experimental protocol on the fNIRS Hitachi ETG-4000 system.
3. Set-up your experimental paradigm. Experimental paradigms can be programmed with different presentation software, including Eprime, Presentation, Psyscope or a Matlab based psychology toolbox. Here we use Matlab based psychology toolbox.
4. Timing is key for data analysis, thus the experimental paradigm must be perfectly timed with data collection. The fNIRS Hitachi ETG-4000 has triggering capabilities, allowing for the experimental paradigm to trigger the data collection or vice versa. Test triggering of presentation program from fNIRS Hitachi ETG-4000. Triggering can be done using parallel, serials, or USB ports. Here we show triggering via the parallel port.
5. Prior to starting the fNIRS study it is important to conduct participant background screening. In the Petitto lab, we conduct background screening by having the participants or their parents fill out study-appropriate standardized questionnaires²⁹.

Participant Arrives

6. It is important to conduct the session and to treat the participants in a professional manner. The participant or the participants' parents/legal guardians must sign a consent form before the experiment begins. It is vital to thank the participant for their time in these important and exciting experiments.
7. The participant is seated comfortably close to the fNIRS testing room. An infant participant may be seated on a parent's lap.

Part 2: Placing Optodes & Using the 10-20 system

Another component of the analysis method that enables consistent data interpretation is the standardization of fNIRS recording protocol. This entails optode placement, participant positioning, and triggering of stimulus presentation software. Both the accurate neuro-anatomical placement of probes and the confirmation of regions of interest (ROIs) are achieved by using the 10–20 system^3,4,30. Further, stereotactic localization of the probe array was confirmed on the participant's skull by overlaying 3D tracking information from a Polhemus Fast trak system onto an anatomical MRI co-registration scan of the participant conducted with vitamin E capsules placed at each probe location^3,4.Optimal participant positioning involved placing participants comfortably in a reclining chair, with fiber optics hanging loosely without contact with the body or chair.

1. The following head measurements are taken with a tape measure and written down on the participants data sheet:
   • Nasion to Inion around
   • Nasion to Inions over top
   • Ear to Ear over top
2. Surgical tape can be used to mark specific target locations. In this experiment we will mark Fp, T3/T4, F8/F7
3. Optode arrays are placed on the participants' head with specific optodes anchored at 10-20 points as directed by the purposes of the experiment.

Part 4: Testing the Optode Array

1. Introduction to the Hitachi ETG-4000 GUI Interface and probe testing.
2. Testing signal: Once optodes are placed on the participants' scalp, the signal quality is tested. If an optode does not have a clear signal, researchers gently remove hair from the connection of the optode and the scalp. On occasion the optodes may need to be wiped with an alcohol swab.

Part 5: Running the experiment.

1. At least two experimenters must be always present in the room; one observing the fNIRS Hitachi ETG-4000 real-time read out and the other observing the participant. Having a video camera focused on participant is highly recommended for post-hoc observations. An advantage of the fNIRS Hitachi ETG-4000 is that the video and fNIRS signal are synched and co-registered. A log containing all relevant information and files generated is kept.
2. There are well-established methods of building experimental hemodynamic paradigms, namely Block design and Event-related designs. For a more complete description please see the recent review paper³¹.

Part 6: Analysis

Once all of the data have been collected, the participant is thanked for their time and willingness to participate and leaves the lab. As analysis is not done on the fNIRS Hitachi ETG-4000, as, instead, the data are exported to an analysis computer.

1. Conversion from μV to hemoglobin concentrations. As raw attenuation values are collected in attenuation of laser strength (as measured in μV), these values must be converted to oxygenated and deoxygenated hemoglobin values. This is done using the modified Beer-Lambert equation.
2. The application of the modified Beer-Lambert is conducted in two steps. Under the assumption that scattering is constant over the path length, first the attenuation for each wavelength (ΔA_λ(_t)) is calculated by comparing the optical density of light intensity during the task (I_task) to the calculated baseline of the signal (I_baseline). The ΔA values for each wavelength and sampled time point (t) to solve the modified Beer-Lambert equation.

   Equation 1

   Equation 2

λ₁∈_deoxy, λ₁∈_oxy, λ₂∈_deoxy and λ ₂∈_oxy are the constants for the extinction coefficients that measure the fraction of light lost to absorption per unit concentration distance in the tissue. The resultant C_deoxy and C_oxy values are the concentrations of deoxygenated and oxygenated hemoglobin for each t.

Part 7: Representative Results

Typical hemodynamic response results in several distinct characteristics. In the oxy-hemoglobin response, there is first a characteristic dip. This dip occurs as a region of neurons activates and depletes available oxygen. As blood flow increases, carrying oxygenated hemoglobin, the oxy-hemoglobin response rises rapidly above the initial baseline levels to a steady state level. When the region is no longer being activated, the oxy-hemoglobin response decays over the next 12-15 seconds and slowly drops back to baseline levels. There is occasionally an undershoot that occurs prior to the hemodynamic response returning to initial baseline levels.

Bad results are usually in the form of optodes not properly seated on the scalp or excessive movement. These types of noise – called, 'Flatling' – are evident in the signal as the microvolt values saturate and a number of different channels, both oxy- and deoxy- response move in a coordinated fashion.

DEMONSTRATION: Shaking the fiber optics.

Statistical Analyses: The extracted oxy and deoxy-hemoglobin values for each channel, for each participant and for each task can then be submitted to conventional statistical analysis, including t-tests, ANOVAs, correlations etc.

In this study, we demonstrated the use of a novel, non-invasive fNIRS brain imaging technology to investigate human brain function in relation to human cognition and perception. fNIRS brain imaging may represent the future of non-invasive brain imaging, particularly with infant and child populations, that may one day be widely available in research labs, physicians' offices, and in the school systems allowing clinicians to apply basic scientific findings about the brain to their clinical practice.