Anna Gerega

Wavelength-resolved measurements of fluorescence lifetime of indocyanine green

We study fluorescence lifetime of indocyanine green (ICG) using femtosecond laser and sensitive detection based on time-correlated single-photon counting. A time-resolved multichannel spectral system is constructed and applied for determination of the fluorescence lifetime of the ICG in different solvents. Emission properties of ICG in water, milk, and 1% intralipid solution are investigated. Fluorescence of the fluorophore of different concentrations (in a range of 1.7-160 μM) dissolved in different solutions is excited by femtosecond pulses generated with the use of Ti:Sa laser tuned within the range of 740-790 nm. It is observed that fluorescence lifetime of ICG in water is 0.166 ± 0.02 ns and does not depend on excitation and emission wavelengths. We also show that for the diffusely scattering solvents (milk and intralipid), the lifetime may depend on the dye concentration (especially for large concentrations of ICG). This effect should be taken into account when analyzing changes in the mean time of arrival of fluorescence photons excited in ICG dissolved in such optically turbid media.

Time-resolved detection of fluorescent light during inflow of ICG to the brain?a methodological study

It was reported that time-resolved reflectance measurements carried out during inflow and washout of an optical contrast agent may provide information on the blood supply to the brain cortex of human adults. It was also shown that a measurement of fluorescence excited in the dye circulating in the brain is feasible. Unfortunately, patterns of time-resolved fluorescence signals observed during in vivo measurements are difficult to interpret. The aim of this study was to analyze the influence of several factors on the fluorescence signals measured during in vivo experiments. A laboratory instrument for recording the distributions of arrival of fluorescence photons was constructed and optimized for measurements on humans. Monte Carlo simulations and laboratory measurements on liquid phantoms as well as in vivo measurements on healthy volunteers were carried out. An influence of source-detector separation, position of the source-detector pair on the head, as well as a dose of the injected indocyanine green (ICG) on the fluorescence signals were studied in detail. It was shown that even for a small dose of ICG (0.025 mg kg(-1)) the time-resolved signals can be successfully detected on the surface of the head. Strong influence of the studied factors on the fluorescence signals was observed. It was also noted that the changes in moments of distributions of arrival times of fluorescence photons depend on the anatomical structure of the tissues located between the source and the detector.

Advantages of fluorescence over diffuse reflectance measurements tested in phantom experiments with dynamic inflow of ICG

Time-resolved measurements of diffuse reflectance and fluorescence were carried out using phantom with dynamic inflow of indocyanine green (ICG) in tubes located at different depths. Better sensitivity of fluorescence signals related to the inflow of the dye was observed in comparison to simultaneously acquired diffuse reflectance. Obtained results can be referred to results of in-vivo measurements. We have observed much larger amplitude of changes in relative number of detected photons, mean time of flight and variance of the distributions of times of arrival of fluorescence photons than amplitudes of respective parameters measured from diffuse reflectance distributions of times of flight of photons. The constructed phantom allows us to study influence of concentration of the dye in the tube and the surrounding medium as well as temporal relation between appearance of the boli in deeper and superficial tube. Results of the study were used in optimization of the time-resolved multichannel system for simultaneous monitoring of fluorescence and reflectance.

Time-resolved multi-channel optical system for assessment of brain oxygenation and perfusion by monitoring of diffuse reflectance and fluorescence

Time-resolved near-infrared spectroscopy is an optical technique which can be applied in tissue oxygenation assessment. In the last decade this method is extensively tested as a potential clinical tool for noninvasive human brain function monitoring and imaging. In the present paper we show construction of an instrument which allows for: (i) estimation of changes in brain tissue oxygenation using two-wavelength spectroscopy approach and (ii) brain perfusion assessment with the use of single-wavelength reflectometry or fluorescence measurements combined with ICG-bolus tracking. A signal processing algorithm based on statistical moments of measured distributions of times of flight of photons is implemented. This data analysis method allows for separation of signals originating from extra- and intracerebral tissue compartments. In this paper we present compact and easily reconfigurable system which can be applied in different types of time-resolved experiments: two-wavelength measurements at 687 and 832 nm, single wavelength reflectance measurements at 760 nm (which is at maximum of ICG absorption spectrum) or fluorescence measurements with excitation at 760 nm. Details of the instrument construction and results of its technical tests are shown. Furthermore, results of in-vivo measurements obtained for various modes of operation of the system are presented.

Assessment of cerebral perfusion in post-traumatic brain injury patients with the use of ICG-bolus tracking method

The aim of this study was to verify the usefulness of the time-resolved optical method utilizing diffusely reflected photons and fluorescence signals combined with intravenous injection of indocyanine green (ICG) in the assessment of brain perfusion in post-traumatic brain injury patients. The distributions of times of flight (DTOFs) of diffusely reflected photons were acquired together with the distributions of times of arrival (DTAs) of fluorescence photons. The data analysis methodology was based on the observation of delays between the signals of statistical moments (number of photons, mean time of flight and variance) of DTOFs and DTAs related to the inflow of ICG to the extra- and intracerebral tissue compartments. Eleven patients with brain hematoma, 15 patients with brain edema and a group of 9 healthy subjects were included in this study. Statistically significant differences between parameters obtained in healthy subjects and patients with brain hematoma and brain edema were observed. The best optical parameter to differentiate patients and control group was variance of the DTOFs or DTAs. Results of the study suggest that time-resolved optical monitoring of inflow of the ICG seems to be a promising tool for detecting cerebral perfusion insufficiencies in critically ill patients.

Variance of time-of-flight distribution is sensitive to cerebral blood flow as demonstrated by ICG bolus-tracking measurements in adult pigs

Variance of time-of-flight distributions have been shown to be more sensitive to cerebral blood flow (CBF) during dynamic-contrast enhanced monitoring of neurotrauma patients than attenuation. What is unknown is the degree to which variance is affected by changes in extracerebral blood flow. Furthermore, the importance of acquiring the arterial input function (AIF) on quantitative analysis of the data is not yet clear. This animal study confirms that variance is both sensitive and specific to changes occurring in the brain when measurements are acquired on the surface of the scalp. Furthermore, when the variance data along with the measured AIF is analyzed using a nonparametric deconvolution method, the recovered change in CBF is in good agreement with CT perfusion values.

Optimization of the method for assessment of brain perfusion in humans using contrast-enhanced reflectometry: multidistance time-resolved measurements

Abstract. The aim of the study was to determine optimal measurement conditions for assessment of brain perfusion with the use of optical contrast agent and time-resolved diffuse reflectometry in the near-infrared wavelength range. The source-detector separation at which the distribution of time of flights (DTOF) of photons provided useful information on the inflow of the contrast agent to the intracerebral brain tissue compartments was determined. Series of Monte Carlo simulations was performed in which the inflow and washout of the dye in extra- and intracerebral tissue compartments was modeled and the DTOFs were obtained at different source-detector separations. Furthermore, tests on diffuse phantoms were carried out using a time-resolved setup allowing the measurement of DTOFs at 16 source-detector separations. Finally, the setup was applied in experiments carried out on the heads of adult volunteers during intravenous injection of indocyanine green. Analysis of statistical moments of the measured DTOFs showed that the source-detector separation of 6 cm is recommended for monitoring of inflow of optical contrast to the intracerebral brain tissue compartments with the use of continuous wave reflectometry, whereas the separation of 4 cm is enough when the higher-order moments of DTOFs are available.

Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult’s brain

Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

Application of optical methods in the monitoring of traumatic brain injury: A review:

We present an overview of the wide range of potential applications of optical methods for monitoring traumatic brain injury. The MEDLINE database was electronically searched with the following search terms: “traumatic brain injury,” “head injury,” or “head trauma,” and “optical methods,” “NIRS,” “near-infrared spectroscopy,” “cerebral oxygenation,” or “cerebral oximetry.” Original reports concerning human subjects published from January 1980 to June 2015 in English were analyzed. Fifty-four studies met our inclusion criteria. Optical methods have been tested for detection of intracranial lesions, monitoring brain oxygenation, assessment of brain perfusion, and evaluation of cerebral autoregulation or intracellular metabolic processes in the brain. Some studies have also examined the applicability of optical methods during the recovery phase of traumatic brain injury . The limitations of currently available optical methods and promising directions of future development are described in this review. Considering the outstanding technical challenges, the limited number of patients studied, and the mixed results and opinions gathered from other reviews on this subject, we believe that optical methods must remain primarily research tools for the present. More studies are needed to gain confidence in the use of these techniques for neuromonitoring of traumatic brain injury patients.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture.