Development of a Near Infrared Multi-Wavelength, Multi-Channel, Time-Resolved Spectrometer for Measuring Brain Tissue Haemodynamics and Metabolism
We present a novel time domain functional near infrared spectroscopy system using a supercontinuum laser allowing us to measure the coefficient of absorption and scattering of up to 16 multiplexed wavelengths in the near infrared region. This is a four detector system that generates up to 3 mW of light for each wavelength with a narrow 2–3 nm FWHM bandwidth between 650 and 890 nm; each measurement of 16 wavelengths per channel can be performed up to a rate of 1 Hz. We can therefore quantify absolute haemoglobin changes in tissue and are currently investigating which and how many wavelengths are needed to resolve additional chromophores in tissue, such as water and the oxidation state of cytochrome-c-oxidase.
KeywordsNIRS TRS Cytochrome-c-oxidase Supercontinuum laser Haemoglobin
Near infrared spectroscopy (NIRS) is commonly used for non-invasive measurements of the concentration changes of oxyhaemoglobin (HbO2) and deoxyhaemoglobin (HHb) in tissue. Typically, continuous wave (CW) systems are used where a reflected/transmitted change in light attenuation through tissue is measured. If the scattering of light in the tissue is assumed constant and the differential path length factor estimated, the modified Beer–Lambert law can be used to calculate changes in chromophore concentrations . CW systems have the benefit of requiring relatively simple and inexpensive components, and can be made into easy to use compact devices.
Time-resolved spectroscopy operates by pulsing short picosecond pulses of light into the tissue through optical fibres. Fast single photon detectors and highly accurate timing electronics are then used to measure the time-of-flight (TOF) of each photon escaping the tissue surface. By repeating this TOF measurement many times a histogram called a temporal point spread function (TPSF) can be generated. We can obtain much more detailed information about the tissue from the TPSF than is possible using a CW technique, including mean path length and the absolute absorption and scattering coefficients .
Advances in technology have reduced the cost and size of the timing electronics needed for TOF measurements, making the technique reasonably accessible. Time-resolved systems are therefore becoming increasingly popular for tissue diagnostics.
In addition to haemoglobin, cytochrome-c-oxidase (CCO) the terminal electron accepter of the respiratory chain is a strong absorber of near infrared light . The absorption spectrum of CCO depends on whether the enzyme is in its oxidised or reduced state; NIRS utilises this to measure the changes in its oxidation state (oxCCO). Although there is a clear optical signature in the difference between the reduced and oxidised forms of CCO, the measurement of oxCCO is considerably more difficult than haemoglobin as the concentration in tissue is of an order of magnitude less . Therefore, in order to decouple the haemoglobin and oxCCO changes accurately it is necessary to enhance the spectroscopic resolution of the NIRS system and measure independently absorption and scattering in many wavelengths . CW broadband  and recently hybrid broadband and frequency domain systems have been used to measure oxCCO . Zhu and colleagues using computational techniques and data from a CW broadband system during severe hypoxic-ischaemia in piglets have found that not only is the number of wavelengths important but there is significant improvement in the estimation of chromophores if specific combinations of wavelengths are used .
In order to address these issues we have designed and built a near infrared time domain multiwavelength spectrometer using a supercontinuum laser source. This enables us to measure the coefficient of absorption (μa) and the reduced coefficient of scattering (μs′) for 16 wavelengths between 650 and 890 nm. Here we describe the hardware of the system, discuss the theory of operation and present some preliminary results from the use of the system to monitor haemodynamic changes in the muscle during an arm cuff occlusion experiment.
2 Instrumentation and Methods
The light is collected by four glass fibre bundles (Loptek) with a diameter of 3 mm and is passed through custom made variable optical attenuators (VOAs) with a range of 0–3.7 OD to four Hamamatsu H7442-50P photomultiplier tube (PMT) modules. As the PMTs have a high gain the VOAs protect against over exposure during the experiment increasing the dynamic range. The signal from the PMTs is passed through a four way router (HRT-41) and the arrival time of each photon is measured with a Becker and Hickl SPC-130-EM time correlated single photon counting card.
The TSPF obtained in time resolved measurements contains information not only from the tissue but also the instrument itself. Therefore, a correction has to be made before the true optical properties of the tissue can be obtained. An instrument response function (IRF, Fig. 24.2a) is recorded before each measurement using neutral density filters (Fig. 24.2c) in order to characterise the factors which contribute towards the broadening of the IRF (laser pulse, optical fibres, photon detectors, and timing electronics).
In order to quantify the optical properties of the tissue, the solution to the diffusion equation for a semi-infinite homogenous medium was convolved with the IRF (Fig. 24.2b) . The convolved model is fitted to the measured TPSF using a non-linear curve fitting function and the absorption coefficient, μa and reduced scattering coefficient, μs′ obtained. The Beer–Lambert law was then used to calculate chromophore concentrations . To test the hardware and theory of operation we performed an arterial cuff-occlusion on the upper arm in one volunteer to induce flow and oxygenation changes in the forearm flexor muscles. The probe was placed on the forearm and measurements were done in reflection mode with source and detector fibres 3 cm apart. After 100 s of baseline measurements we inflated the cuff at 200 mmHg for 300 s, following cuff deflation we continue monitoring the muscle recovery for 5 min.
Data were collected every second for eight common wavelengths used in near infrared spectroscopy [690 750 761 790 801 834 850 870]. The average count rate over the experiment was kept at over 106/s to provide a good enough SNR for each wavelength. The diffusion equation model was then fitted to each TPSF to resolve absorption and scattering.
We have developed a four-channel NIR time-resolved spectrometer using a supercontinuum laser source and tunable narrow band filter system capable of measuring the TPSFs of 16 wavelengths between 650 and 890 nm every second in order to quantify the scattering and absorption independently for tissue. This offers us the ability to extract changes in haemoglobin and other chromophores in tissue such as CCO. We have presented preliminary results of the operation of the system for one channel and eight wavelengths during an arm cuff occlusion test. We are currently using the system to investigate wavelength selection optimisation for resolving HbO2, HHb and oxCCO and will be carrying out a series of functional activation studies.
The authors would like to thank The Wellcome Trust (088429/Z/09/Z) for the financial support of this work.
- 8.T. Zhu et al (2012) Optimal wavelength combinations for resolving in-vivo changes of haemoglobin and cytochrome-c-oxidase concentrations with NIRS. Biomed Optics and 3-D Imaging, OSA Technical Digest. JM3A.6Google Scholar
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