Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb + Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R = 0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb + Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF] = 2.7-2.8) in a living heart tissue.     

Differential pathlength factor for diffuse photon scattering through tissue by a pulse-response method.

Although near-infrared (NIR) spectroscopy may one day provide a noninvasive measurement of oxidative metabolism in tissue, the method cannot be fully quantitative until the mean pathlength traveled by photons between reference and output detectors (i.e, optrodes) is known. In NIR spectroscopy, photons are transported primarily by diffuse scattering, and their mean pathlength can be expressed by a differential path factor (DPF) whose value is greater than the interoptrode distance. Beginning with a P1 diffusion approximation of the Boltzmann equation, one-dimensional photon currents originating from plane, line, and point photon sources were analyzed. DPF was formulated from the attenuation of light intensity generated by constant sources, and an equation for the mean time of flight of photons between reference and output optrodes, delta tau, was derived for arbitrarily pulsed sources. The results indicate that (1) the attenuation of light in tissue does not, in general, vary with interoptrode distance in the manner predicted by Beer's law; (2) the relationship between DPF and interoptrode distance is nonlinear and geometry-dependent; and (3) in spite of these nonidealities, DPF is equal to the product of delta tau and the speed of light.     

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.     

Measurement of cerebral blood volume using near-infrared spectroscopy and indocyanine green elimination.

Methods for measuring cerebral blood volume (CBV) have traditionally used radioisotopes. More recently, near-infrared spectroscopy (NIRS) has been used to measure CBV by using a technique involving O(2) desaturation of cerebral tissue, where the observed change in the concentration of oxygenated hemoglobin is a marker of the volume of blood contained within the brain. A new integration method employing NIRS is described by using indocyanine green (ICG) as the intravascular marker. After bolus injection, concentration-time integrals of cerebral tissue ICG concentration ([ICG](tissue)) measured by NIRS are compared with corresponding integrals of the cerebral blood ICG concentrations ([ICG](blood)) estimated by high-performance liquid chromatography of peripheral blood samples with allowance for cerebral-to-large-vessel hematocrit ratio. It is shown that CBV = integral [ICG]tissue/[ICG]blood. Measurements in 10 adult volunteers gave a mean value of 1.1 +/- 0.39 (SD) ml/100 g illuminated tissue. This result, although lower than previous NIRS estimations, is consistent with the long extracerebral path of light in the adult head. Scaling of results is required to take into account this component of the optical pathlength.     

Noninvasive quantitative analysis of blood oxygenation in rat skeletal muscle

Using the isolated perfused rat hindlimb and the fluorocarbon-transfused rat, we have examined the optical characteristics of the rat skeletal muscle in the near-infrared region. The total contribution of myoglobin and cytochromes to the overall absorbance change was less than 10%. Analyzing transmitted light at 700, 730, and 805 nm, we found linear relationships between the absorbance and the hemoglobin concentrations at hematocrit values from 15 to 50% in the inflowing perfusate. Based on the relationship, we determined the ratio of absorption coefficients at 700, 730, and 805 nm of oxy- and deoxy-hemoglobins of blood in the thigh muscle. The values in thigh muscle were significantly smaller than those in hemoglobin solutions for deoxygenated blood. On the other hand, the values in thigh muscle were larger than those in hemoglobin solutions for oxygenated blood. Solving simultaneous equations by the use of these absorption coefficients, we calculated the changes in the contents of oxy-, deoxy-, and total hemoglobins in the anesthetized rat hindlimb under various conditions. The oxygen saturation of blood determined by our optical method in the thigh muscle was very close to that in the vena cava measured directly with a gas analyzer.     

Photonics‐based In Vivo total hemoglobin monitoring and clinical relevance

Anemia is a serious disorder which, as a result of antiquated invasive blood testing, is undiagnosed in millions of people in the U.S. As a result of the clinical need, many technological solutions have been proposed to measure total blood hemoglobin, and thus diagnose anemia, noninvasively. Because hemoglobin is the strongest chromophore in tissue, spectroscopic methods have been the most prevalently investigated. Difficulties in extracting a quantitative estimation of hemoglobin based on tissue absorption include variability in the absorption spectra of hemoglobin derivatives, interference from other tissue chromophores, and interpatient physiological variations affecting the effective optical pathlength of light propagating in tissue. In spite of these challenges, studies with a high degree of correlation between in vitro and in vivo measured total hemoglobin have been disclosed using variants of transmission and diffuse reflection spectroscopy in assorted physiological locations. A review of these technologies and the relevant advantages/disadvantages are presented here. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.     

Laser spectroscopic assessment of induced hyperoxia--an animal experiment in lambs

The noninvasive determination of biochemical parameters has become an important aspect of intensive care medicine. The newly developed monitors for laser reflectometry provide the possibility of spectroscopic monitoring. The equipment consists of a near-infrared data collection unit and a personal computer. The four laser diodes emit light at wavelengths of 775, 805, 845 and 904 nm. By analyzing the changes in optical density during laser irradiation of biological tissue, information is obtained about the relative changes in the concentration of hemoglobin and the blood volume. In animal experiments with ten fetal lambs we evaluated the reliability of near infrared laser spectroscopy. Fetal hyperoxia was achieved by means of an extracorporeal circuit with interposition of a membrane oxygenator (0.8 m2, Scimed). During the induced hyperoxia the laser spectroscopic tracings showed a rise in the HbO2 signal with a synchronous decrease in the HbR signal. Additionally, the spectroscopic pattern showed a characteristic initial rise in the intracerebral blood volume, which stabilized after 4 minutes. We found a significant correlation between the intermittently measured PO2 values of the arterial blood samples and the laser spectroscopic HbO2 and HbR signals (r = 0.87, and r = -0.82, respectively; p less than 0.001). Furthermore, hyperoxia was indicated by the laser system with a short lag time. We conclude that laser spectroscopy is a reliable method with a high potential for clinical routine use in intensive care, as it provides noninvasive continuous information at comparatively low costs using portable monitors.     

A Numerical Simulation of the Diffusion of Near Infrared Light Through Brain Tissue

A numerical simulation of the transport of light energy in the near infrared region of the spectrum through human brain tissue is presented. This simulation models the use of near infrared spectroscopic techniques to quantify the levels of oxygen present in brain tissue. Successful application of the technique requires knowledge of the optical pathlength in the tissue, and it is the goal of this simulation to quantify the relationship of the optical pathlength and the oxygenation state of the tissue. Both implicit and explicit finite element schemes for unstructured grids are implemented and discussed. Several application simulations using three tissue grids of varying degrees of physiological accuracy are then conducted, and figures for the optical pathlength of light through the tissue at varying levels of oxygenation are computed. These results are then used to develop a quantitative relationship between the pathlength and the absorption parameter for the three tissue models which we explore.     

A Numerical Simulation of the Diffusion of Near Infrared Light Through Brain Tissue

A numerical simulation of the transport of light energy in the near infrared region of the spectrum through human brain tissue is presented. This simulation models the use of near infrared spectroscopic techniques to quantify the levels of oxygen present in brain tissue. Successful application of the technique requires knowledge of the optical pathlength in the tissue, and it is the goal of this simulation to quantify the relationship of the optical pathlength and the oxygenation state of the tissue. Both implicit and explicit finite element schemes for unstructured grids are implemented and discussed. Several application simulations using three tissue grids of varying degrees of physiological accuracy are then conducted, and figures for the optical pathlength of light through the tissue at varying levels of oxygenation are computed. These results are then used to develop a quantitative relationship between the pathlength and the absorption parameter for the three tissue models which we explore.     

Clearance and tissue distribution of functionalized polymeric liposomes from the blood stream of rats

Polymeric liposomes containing a synthetic porphinato-iron-imidazole complex (hemoglobin or red blood cell model) were labeled by introducing 1,2-di[1-14C]palmitoyl-sn-glycero-3-phosphocholine into their polymerized bilayers. After intravenous injection into rats, their clearance from a blood stream was measured. The apparent half-life time (50% disappearance time) was about 14 +/- 2 h. Their tissue distribution was determined with time by whole autoradiographic measurement.     

Simple delay monitor for droplet sorters

We have constructed a simple device by which the optimal delay time between optical measurement of a cell and the application of the droplet charging pulse can be determined directly in a flow sorter. The device consists of a stainless steel chamber in which the sorted droplets are collected. In the collection chamber the collected droplets run through a capillary where a continuous fluorescence measurement is made. With a sample of fluorescent particles, the delay time is optimal when the measured fluorescence is maximal. The measuring volume is always filled with the last droplets sorted (about 3,000). With this device, the setting of the delay time can be done in a few seconds without the need for microscopical verification. The fluorescence in the collection chamber is excited and detected via optical fibers using about 10% of the light of the existing laser from the flow cytometer and an extra photomultiplier.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 mus duration dye laser flash of varying intensities as an excitation source. The yield (phi) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectoscopy at 515 nm, while the yield phi following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from phi utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (greater than 10(16) photon . cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 mus after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a single-triplet bimolecular rate constant of gammast = 10(-8) cm3 . s-1, the following observations can be accounted for: (1) the rapid initial drop in phi and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Exciton Annihilation in the Two Photosystems in Chloroplasts at 100°K

The fluorescence yield (F) of spinach chloroplasts at 100°K measured at 735 nm (photosystem I fluorescence—F 735) and at 685 nm (photosystem II fluorescence—F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-μs pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10¹⁵ to 10¹⁸ photons/cm² per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.     

Investigation of Early Plasma Evolution Induced by Ultrashort Laser Pulses

Early plasma is generated owing to high intensity laser irradiation of target and the subsequent target material ionization. Its dynamics plays a significant role in laser-material interaction, especially in the air environment^1-11.Early plasma evolution has been captured through pump-probe shadowgraphy^1-3 and interferometry^1,4-7. However, the studied time frames and applied laser parameter ranges are limited. For example, direct examinations of plasma front locations and electron number densities within a delay time of 100 picosecond (ps) with respect to the laser pulse peak are still very few, especially for the ultrashort pulse of a duration around 100 femtosecond (fs) and a low power density around 10¹⁴ W/cm². Early plasma generated under these conditions has only been captured recently with high temporal and spatial resolutions¹². The detailed setup strategy and procedures of this high precision measurement will be illustrated in this paper. The rationale of the measurement is optical pump-probe shadowgraphy: one ultrashort laser pulse is split to a pump pulse and a probe pulse, while the delay time between them can be adjusted by changing their beam path lengths. The pump pulse ablates the target and generates the early plasma, and the probe pulse propagates through the plasma region and detects the non-uniformity of electron number density. In addition, animations are generated using the calculated results from the simulation model of Ref. ¹² to illustrate the plasma formation and evolution with a very high resolution (0.04 ~ 1 ps).Both the experimental method and the simulation method can be applied to a broad range of time frames and laser parameters. These methods can be used to examine the early plasma generated not only from metals, but also from semiconductors and insulators.     

Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.

Ultraviolet (UV) irradiation can initiate complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future'. In this review we characterize laser-induced crosslinking and explore the applications of this method.     

A quantitative method for the analysis of glycated and glutathionylated hemoglobin by matrix-assisted laser desorption ionization-time of flight mass spectrometry

The quantization of glycated isoforms of hemoglobin has been increasingly used in clinical practice in recent years. Glycated hemoglobin is currently considered the most important measurement for long-term control of the glycemic state and it has become a reference tool for the management of diabetes. Glutathionylated hemoglobin is an increasingly clinically relevant covalent adduct of glutathione with beta chain of the globin and its concentration has been correlated with oxidative stress. We have developed an innovative technique based on linear mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for quantitative analysis of hemoglobin species. This method was applied to the quantification of glycated and glutathionylated hemoglobin. A rigorous comparison was pursued to evaluate the analytical performances in quantifying glycated hemoglobin in comparison to an established high-performance liquid chromatography method. Our results indicated a complete equivalence between the two methods. The same analysis enabled the quantitative determination of the glutathionylated hemoglobin fraction. This isoform was investigated in an adult Italian population (184 individuals, 101 males and 83 females), indicating a bimodal distribution of this species. In fact 65.22% of screened individuals had glutathionylated hemoglobin levels lower than 0.50% while 34.78% had glutathionylated hemoglobin levels higher than 0.50%. A semiautomatic robotic procedure was developed for fast analysis of a large number of samples. This is the first report of a quantitative application of linear MALDI-TOF mass spectrometry for the determination of glutathionylated hemoglobin in blood samples. This method allows fast screening of this hemoglobin isoform, therefore opening the route to explore its specificity and sensitivity as a molecular biomarker.     

Temperature regime of biological tissue under photodynamic therapy

An analytical model is proposed to calculate heating of human skin cover under laser light action of photodynamic therapy. A photosensitizer of "Fotolon" is taken as an example. Temperatures of skin surface and of deep dermis regions are studied as a function of time under pulsed and stationary irradiation of skin surface at the wavelength of 665 nm corresponding to the maximum of the photosensitizer absorption band. It is shown that, under the action of a short light pulse, the photosensitizer can lead to an essential temperature rise of dermis due to a considerable increase in its absorption coefficient. However, this rise does not destruct tissue cells because of the short action. Under stationary irradiation, the photosensitizer concentration has a low effect on the temperature regime of tissue. This is related with the specific features in heating of the medium by red light, where the main thermal process in skin is heat transfer over tissue volume from epidermis having a substantially larger absorption coefficient than that of dermis in the said spectral range. The role of blood perfusion in dermis and its effect on the temperature regime of tissue are evaluated.     

Temperature regime of biological tissue under photodynamic therapy

An analytical model is proposed to calculate heating of human skin cover under laser light action of photodynamic therapy. A photosensitizer of «Fotolon» is taken as an example. Temperatures of skin surface and of deep dermis regions are studied as a function of time under pulsed and stationary irradiation of skin surface at the wavelength of 665 nm corresponding to the maximum of the photosensitizer absorption band. It is shown that, under the action of a short light pulse, the photosensitizer can lead to an essential temperature rise of dermis due to a considerable increase in its absorption coefficient. However, this rise does not destruct tissue cells because of the short action. Under stationary irradiation, the photosensitizer concentration has a low effect on the temperature regime of tissue. This is related with the specific features in heating of the medium by red light, where the main thermal process in skin is heat transfer over tissue volume from epidermis having a substantially larger absorption coefficient than that of dermis in the said spectral range. The role of blood perfusion in dermis and its effect on the temperature regime of tissue are evaluated.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5–4.0 μs duration dye laser flash of varying intensities as an excitation source. The yield (Ф) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectroscopy at 515 nm, while the yield Ф following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10–12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from Ф utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (10¹⁶ photon · cm⁻²) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1–2 μs after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules.

At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse.

A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a singlet-triplet bimolecular rate constant of γ_st = 10⁻⁸ cm³ · s⁻¹, the following observations can be accounted for: (1) the rapid initial drop in Ф and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.