Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.     

Measurement of tumor oxygenation using new frequency domain phosphorometers

Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.     

In vivo oxygen imaging using green fluorescent protein

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs. heart; ischemia; hypoxia; molecular imaging     

Fringe mode transmittance laser Doppler microscope anemometer: its adaptation for measurement in the microcirculation

Blood flow analysis in the microcirculation requires accurate measurement of velocity, volume flow and shear-rate versus shear-stress relationships. The resolution of most anemometers is too limited to obtain useful measurements, especially near the blood vessel wall and at branches and bifurcations. To make such measurements possible with a noninvasive, high resolution, accurate technique, we have developed a fringe mode, transmittance laser Doppler microscope anemometer (LDMA). This system has an intrinsically high spatial resolution (10 × 12 μm), and does not require a high concentration (10⁶/cm³) of scatterers or red blood cells (RBC) as in our application. Preliminary measurements of water flow in a rectangular channel were conducted to ascertain the reliability and accuracy of velocity measurements using the LDMA. Velocity profiles were then measured by the LDMA system in arterioles 38–135 μm in diameter, in the transparent, everted cheek pouch of the anaesthelized hamster. The extremely high resolution of the optical system, and the ultra-fine traversing mechanism of the microscope slage, made velocity readings larger than 0.02 mm/s with accuracy and reproducibility better than 1%, possible near the wall to within 7–10 μm.     

PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on Po2 in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses Po2 in a motionless tissue, but less in flowing blood, creating an apparent transmural Po2 drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal Po2 gradients in arterioles, a Po2 rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low Po2 in lymphatic microvessels, and both low and uniform tissue Po2. This alternative explanation for reported paradoxical results of Po2 distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free Po2 measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic Po2 measurements in the microcirculation using the PQM.     

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain''s ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg¹. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 μm. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.     

Quantitative analysis of the hemoglobin oxygenation state of rat brain in vivo by picosecond time-resolved spectrophotometry

Through the use of a picosecond laser pulse of near-infrared light at 1,064 nm, the temporal profile of the transmitted light through the anesthetized rat head has been investigated. The light intensity at a certain time after the input pulse was exponentially attenuated by the hemoglobin concentration with hematocrit values from 1.5 to 50%, although the transmitted pulse broadened markedly due to scattering by the cerebral tissue. The optical pathlength, which is required for quantitation of the absolute absorbance change, was directly determined, by the time of flight measurement of the light pulses, as the product of the velocity of light in tissue and time. The mean concentration of hemoglobin in the brain could be determined quantitatively by the use of this pathlength. The oxygen saturation of venous blood determined by our time of flight measurement was very close to that in the internal jugular vein determined directly with a gas analyzer. Thus, the picosecond laser technique is useful for quantifying the blood oxygenation in tissues.     

Fiberoptic laser-doppler anemometer microscope applied to the cerebral microcirculation in rats

We have applied our developed fiber-optic laser-Doppler anemometer microscope (FLDAM) for the study of the cerebral microcirculation in the rat. The red cell velocity in single pial microvessels was successfully measured through a closed cranial window for the vessel diameter range from 7.8 to 230 μm. The temporal resolution of the FLDAM was sufficiently high to detect the pulsation in the arterioles. Arterio-venous distributions of the temporal mean red cell velocity and wall shear rate are also described.     

Direct evidence of the vasodilator action of carbon dioxide on subcutaneous microvasculature in rats by use of intra-vital video-microscopy

The therapeutic effects of carbon dioxide (CO₂) on cutaneous tissue blood flow in the human have long been well recognized. Although CO₂ has vasodilator action, in-vivo evidence of its action on the microcirculation of the skin, and of its mechanism, has rarely been reported. We studied the direct effects of CO₂ on in-vivo microvasculature and blood flow rate by using an intra-vital video-microscopic system. Brown Norway rats were anesthetized by intraperitoneal administration of alpha-chloralose and urethane. In order to measure inner diameter and red blood cell velocity (Vrbc) for a microvessel, the dorsal skin window was draped on an observation box placed inside a bath. Vrbc was derived from the cross-correlation function of paired segments of dual-window intensity in the video of microvascular images of the subcutaneous tissue. We measured pH in subcutaneous tissue by making a dorsal skin tube. After topical application of CO₂ dissolved in water via the skin of the rat, we observed both vasodilatation and an increase in blood flow of the micro vessels. The pH of subcutaneous tissue also decreased after CO₂ application. The CO₂ reduced the pH of subcutaneous tissue and inhibited vascular smooth muscle contraction, resulting in dilatation of the vasculature of the skin microcirculation.     

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer''s disease, and head injury.Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.Click here to view the related article Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.Download video file.^{(53M, mov)}     

Measurement of Local Partial Pressure of Oxygen in the Brain Tissue under Normoxia and Epilepsy with Phosphorescence Lifetime Microscopy

In this work a method for measuring brain oxygen partial pressure with confocal phosphorescence lifetime microscopy system is reported. When used in conjunction with a dendritic phosphorescent probe, Oxyphor G4, this system enabled minimally invasive measurements of oxygen partial pressure (pO2) in cerebral tissue with high spatial and temporal resolution during 4-AP induced epileptic seizures. Investigating epileptic events, we characterized the spatio-temporal distribution of the "initial dip" in pO2 near the probe injection site and along nearby arterioles. Our results reveal a correlation between the percent change in the pO2 signal during the "initial dip" and the duration of seizure-like activity, which can help localize the epileptic focus and predict the length of seizure.     

Early recovery of microvascular perfusion induced by t-PA in combination with abciximab or eptifibatide during postischemic reperfusion

Background  

GPIIb/IIIa inhibitors abciximab and eptifibatide have been shown to inhibit platelet aggregation in ischemic heart disease. Our aim was to test the efficacy of abiciximab (Reo Pro) or eptifibatide (Integrilin) alone or in combination with plasminogen activator (t-PA) in an experimental model of ischemia reperfusion (I/R) in hamster cheek pouch microcirculation visualized by fluorescence microscopy. Hamsters were treated with saline, or abiciximab or eptifibatide or these drugs combined with t-PA infused intravenously 10 minutes before ischemia and through reperfusion. We measured the microvessel diameter changes, the arteriolar red blood cell (RBC) velocity, the increase in permeability, the perfused capillary length (PCL), and the platelet and leukocyte adhesion on microvessels.     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorimeters for analysis of decay profiles and real time monitoring of exponential decay and oxygen concentrations

A phosphorimeter which can be assembled at low cost from mainly commercially available components and which has better time resolution, data acquisition rate, sensitivity, and flexibility than commercially available instruments is described. As a phosphorescence analyzer the instrument can measure phosphorescence lifetimes ranging from approximately 30 microseconds to seconds from samples with variable intensity, excitation, and emission spectra and which may follow complex decay behavior. Configured as a phosphorescence monitor it is designed for fast, repetitive calculation of phosphorescence lifetime, assuming single-exponential decay, and can be used to calculate oxygen concentration in biological samples in real time.     

Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

Microcirculatory hematocrit and blood flow

Direct measurements from many laboratories indicate that the oxygen tension in skeletal muscle is significantly less than in the large veins draining these tissues. Harris (1986) has proposed that because of the parallel anatomic arrangement of large arterioles and venules in skeletal muscle, a counter-current exchange between these vessels can occur. He theorized that diffusion of O2 between arteriole and venule would lower the PO2 in the blood as it enters capillaries and result in a decreased tissue PO2 and an increase in large vein PO2. Calculations (Appendix) show that the amount of O2 transferred between arteriole and venule is inadequate to account for this difference in PO2 between tissue and veins due to the small surface area that is involved. It is well documented that the microcirculatory hematocrit ranges between 20 and 50% of that in the supply vessels. The reduced hematocrit lowers the oxygen content in these vessels and results in a low oxygen tension in the surrounding tissue. True arteriovenous shunts are not present in most skeletal muscles, but 15-20% of the microvessels represent thoroughfare or preferential flow channels. It is suggested that these vessels contain a greater than normal hematocrit to account for a conservation of red cell mass across the microcirculation. Furthermore, it is shown that the hematocrit in the preferential flow channels is an inverse function of the flow rate for any level of the microcirculatory hematocrit. The increased hematocrit raises the flow resistance in these vessels which reduces flow further and represents a positive feedback condition which may contribute to the intermittent and uneven flow patterns which are present within the microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microvascular and mitochondrial PO2 simultaneously measured by oxygen‐dependent delayed luminescence

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO₂ values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO₂ (μPO₂) and mitochondrial PO₂ (mitoPO₂) in rats. The μPO₂ measurements are based on oxygen‐dependent quenching of phosphorescence of the near‐infrared phosphor Oxyphor G2. The mitoPO₂ measurements are based on oxygen‐dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber‐based time‐domain setup consisting of a tunable pulsed laser, 2 red‐sensitive gated photomultiplier tubes and a simultaneous sampling data‐acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous μPO₂ and mitoPO₂ measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)