Our lab develops light-based tools that provide diagnostic information for maternal health applications. We often use optical spectroscopy because of the plethora of health-related information it can provide non-invasively, such as monitoring blood oxygenation and hemoglobin concentration, both of which are important physiologic parameters to monitor during pregnancy. Some solvents in particulate water have strong absorption that can overshadow the solutes of interest.
Further, spectra-spanning the VIS-SWIR region often have large differences in absorption at different wavelength ranges, and we have to change experimental settings for different regions to obtain high SNL spectra. Difficulty in acquiring VIS-SWIR biological spectra has contributed to limited published biological absorption spectra, and we aim to simplify this process. In addition, an improved understanding of each biological component's specific spectral characteristics will help choose optimized wavelengths that could minimize skin pigmentation bias caused by strong melanin absorption.
We hope our protocol will enable researchers to expand the current VIS-SWIR library of biological absorbers. These results also highlight the impact of melanin on optical devices as a function of wavelength and can be used to design systems with minimal skin pigmentation bias, such as in the SWIR. To prepare the oxygenated hemoglobin sample with whole heparinized human blood, pipette 1.8 milliliters of blood into a micro centrifuge tube.
Centrifuge the tube at 9.6g for 10 minutes, and discard approximately 850 microliters of supernatant without disturbing the pellet. Reconstitute the pellet in approximately 850 microliters of deuterated water, and centrifuge again at 9.6g for 10 minutes. After the last centrifugation, to lyse red blood cells, reconstitute the red blood cell pellets with 4.5 milliliters of deuterated water.
If the pellet is adhering to the tube, rinse with a portion of deuterated water to loosen it. Using a blunt tip needle and syringe, remove the lysed blood solution from the tube. Then detach the blunt tip needle from the syringe, and discard it in a biohazard sharps container.
Next, attach a 0.22-micrometer syringe filter to the syringe, and filter the contents into a 10-millimeter path length, 3.5 milliliter glass, or a quartz cuvette. Secure the cuvette with an airtight stopper to prevent contamination. Wrap Parafilm around the stopper to ensure a completely airtight seal.
Prepare a reference by filling a clean cuvette with the same path, length, and volume, with deuterated water for baseline and reference measurements. To prepare the deoxygenated hemoglobin sample, add sodium dithionite to the blood solution in the cuvette. Secure the cuvette with an airtight stopper and wrap Parafilm around the stopper to ensure an airtight seal.
Carefully tilt the cuvette from side to side until the sodium dithionite is completely dissolved. Observe the color change of the blood solution from red to a darker purple red. To begin, turn on the spectrometer lamp and detectors, allowing the system to warm up for 5 to 20 minutes.
Clean the cuvettes with ethanol using a Kimwipe. For a dual-beam spectrometer, place cuvettes filled with deuterated water as the reference solution in both the sample and reference chambers. Select the appropriate wavelength range and the detector based on the spectral region to be measured.
Obtain reference measurements using the default parameters. Next, replace the cuvette in the sample chamber with the cuvette containing the oxygenated hemoglobin. Switch to Live Mode to view absorbance continuously as measurement parameters are tuned.
Starting with default spectrometer parameters, adjust the reads per datum and bandwidth to obtain a qualitatively clean absorption spectrum without saturation. If modifying the incident light power, bandwidth, exposure time, and reads per datum does not result in a high signal-to-noise ratio spectrum, change the sample concentration, placing the reference cuvette in either the sample or reference chamber or the path length of the sample and the reference to obtain a high signal-to-noise ratio spectrum. Once the settings are optimized for the sample, document the settings for each spectrum acquired.
Using the optimized settings, remeasure using the same settings with the reference solution in the reference and sample chambers to obtain the optimized reference measurement. Next, replace the reference cuvette in the sample chamber with the oxygenated hemoglobin sample cuvette, and take an optimized sample measurement. Save both the reference and sample measurements separately.
Using the following equation, calculate the absorbance A by treating the reference measurement as I0 and the sample measurement as I.For post-processing, open data analysis software such as MATLAB, and load the absorbance measurements acquired from different regions of the spectrum. Apply a multiplication factor to one spectral region to stitch together different regions with varying sample concentrations or path lengths. The oxygenated hemoglobin spectrum showed a strong correlation with a published spectrum by Prahl, with distinct absorption peaks at 415 nanometers and a doublet between 540 to 575 nanometers in the visible region, along with a broad absorption feature between 800 and 1, 100 nanometers in the near infrared.
In the short wave infrared region. The oxygenated hemoglobin spectrum showed low absorption, likely due to residual water content. The deoxygenated hemoglobin spectrum displayed a strong correlation with Prahl's spectrum, featuring prominent peaks at 430 nanometers, 560 nanometers, and 760 nanometers, and smaller peaks around 1, 200 nanometers, and between 1, 400 to 1, 600 nanometers.
