Alexey Aprelev

Oxygen Microscopy by Two‐Photon‐Excited Phosphorescence

High-resolution images of oxygen distributions in microheterogeneous samples are obtained by two-photon laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO(2) in cells.

Multifunctional magnetic rotator for micro and nanorheological studies.

We report on the development of a multifunctional magnetic rotator that has been built and used during the last five years by two groups from Clemson and Drexel Universities studying the rheological properties of microdroplets. This magnetic rotator allows one to generate rotating magnetic fields in a broad frequency band, from hertz to tens kilohertz. We illustrate its flexibility and robustness by conducting the rheological studies of simple and polymeric fluids at the nano and microscale. First we reproduce a temperature-dependent viscosity of a synthetic oil used as a viscosity standard. Magnetic rotational spectroscopy with suspended nickel nanorods was used in these studies. As a second example, we converted the magnetic rotator into a pump with precise controlled flow modulation. Using multiwalled carbon nanotubes, we were able to estimate the shear modulus of sickle hemoglobin polymer. We believe that this multifunctional magnetic system will be useful not only for micro and nanorheological studies, but it will find much broader applications requiring remote controlled manipulation of micro and nanoobjects.

The Growth of Sickle Hemoglobin Polymers

The measurement of polymer growth is an essential element in characterization of assembly. We have developed a precise method of measuring the growth of sickle hemoglobin polymers by observing the time required for polymers to traverse a photolytically produced channel between a region in which polymers are created and a detection region. The presence of the polymer is functionally detected by observing its ability to create new polymers through the well-established process of heterogeneous nucleation. Using this method, we have determined the rate constants for monomer addition to and release from polymer ends, as well as their temperature dependences. At 25°C we find k(+) = 84 ± 2 mM⁻¹ s⁻¹ and k(-) = 790 ± 80 molecules/s from each end. These numbers are in accord with differential interference contrast measurements, and their ratio gives a solubility measured on individual fibers. The single-fiber solubility agrees with that measured in sedimentation experiments. The concentration dependence of the monomer addition rate is consistent with monomer addition, but not oligomer addition, to growing polymers. The concentration dependence suggests the presence of an activation enthalpy barrier, and the rate of monomer addition is not diffusion-limited. Analysis of the temperature dependence of the monomer addition rate reveals an apparent activation energy of 9.1 ± 0.6 kcal/mol.

The microrheology of sickle hemoglobin gels.

Sickle cell disease is a rheological disease, yet no quantitative rheological data exist on microscopic samples at physiological concentrations. We have developed a novel method for measuring the microrheology of sickle hemoglobin gels, based on magnetically driven compression of 5- to 8-microm-thick emulsions containing hemoglobin droplets approximately 80 microm in diameter. Using our method, by observing the expansion of the droplet area as the emulsion is compressed, we were able to resolve changes in thickness of a few nanometers with temporal resolution of milliseconds. Gels were formed at various initial concentrations and temperatures and with different internal domain structure. All behaved as Hookean springs with Youngs modulus from 300 to 1500 kPa for gels with polymerized hemoglobin concentration from 6 g/dl to 12 g/dl. For uniform, multidomain gels, Youngs modulus mainly depended on the terminal concentration of the gel rather than the conditions of formation. A simple model reproduced the quadratic dependence of the Youngs modulus on the concentration of polymerized hemoglobin. Partially desaturated samples also displayed quadratic concentration dependence but with a smaller proportionality coefficient, as did samples that were desaturated in steps; such samples were significantly less rigid than gels formed all at once. The magnitude of the Youngs modulus provides quantitative support for the dominant models of sickle pathophysiology.

Universal Metastability of Sickle Hemoglobin Polymerization

Sickle hemoglobin (HbS) polymerization occurs when the concentration of deoxyHbS exceeds a well-defined solubility. In experiments using sickle hemoglobin droplets suspended in oil, it has been shown that when polymerization ceases the monomer concentration is above equilibrium solubility. We find that the final concentration in uniform bulk solutions (i.e., with negligible boundaries) agrees with the droplet measurements, and both exceed the expected solubility. To measure hemoglobin in uniform solutions, we used modulated excitation of trace amounts of CO in gels of HbS. In this method, a small amount of CO is introduced to a spatially uniform deoxyHb sample, so that less than 2% of the sample is liganded. The liganded fraction is photolyzed repeatedly and the rate of recombination allows the concentration of deoxyHbS in the solution phase to be determined, even if polymers have formed. Both uniform and droplet samples exhibit the same quantitative behavior, exceeding solubility by an amount that depends on the initial concentration of the sample, as well as conditions under which the gel was formed. We hypothesize that the early termination of polymerization is due to the obstruction in polymer growth, which is consistent with the observation that pressing on slides lowers the final monomer concentration, making it closer to solubility. The thermodynamic solubility in free solution is thus achieved only in conditions with low polymer density or under external forces (such as found in sedimentation) that disrupt polymers. Since we find that only about 67% of the expected polymer mass forms, this result will impact any analysis predicated on predicting the polymer fraction in a given experiment.

The Physical Foundation of Vasoocclusion in Sickle Cell Disease

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.

Calibrating Sickle Cell Disease

Sickle cell disease is fundamentally a kinetic disorder, in which cells containing the mutated hemoglobin (hemoglobin S; HbS) will cause occlusion if they sickle in the microvasculature, but have minimal (or no) consequences if they sickle in the venous return. Physiologically, sickling always occurs when some ligands are present; nonetheless, the kinetics in the presence of ligands are virtually unstudied. Sickling arises from nucleation-controlled polymer formation, triggered when the HbS loses ligands (e.g., oxygen). Thus, understanding how nucleation responds to the presence of oxygen is the key to understanding how sickling proceeds in a physiological context. We have measured the rate of nucleus formation in HbS partially liganded with NO or CO, which we find have equivalent effects in reducing the nucleation rates. We find that hemoglobin must be in the T (tense) quaternary structure for nucleation, but the presence of ligands inhibits nucleus formation even when the correct quaternary structure is present. From these results, we can predict the fraction of cells that will sickle at any given partial ligand saturations. The ability to make such predictions may prove especially useful in designing future therapies, particularly those where the oxygen affinity is perturbed.

Note: Professional grade microfluidics fabricated simply

Microfluidics has found increasingly wide usage in the research and teaching laboratory, but setting up a facility for its production has typically required either significant capital expense or sacrifice of quality. We present an approach to produce devices, without a clean room, using LEDs and spin-coaters, and plasma bonded using a commercial microwave oven. Submicron features can be readily reproduced with good fidelity.

Diagnosing Sickle Cell Disease

Sickle cell disease is a world-wide problem, and its diagnosis remains a challenge in both developed and underdeveloped nations. In Africa, for example, where 300,000 children are born each year with the disease, early identification and elementary care could propel the lifespan from the current 5 years to near the 40 years seen in places such as the US. In places where screening is more nearly universal, issues of diagnosis are more likely to arise when emergency care is required and a patients sickle cell status is uncertain. Moreover, the possibilities of mass migrations can again confound issues of patient identification that might be simple in a geographically fixed population. We are accordingly developing rapid and inexpensive tests for sickle cell disease. Our most recent device employs a narrow (100 micron) tube, which will draw in blood based on capillary forces. When sickle blood is deoxygenated blood, it will rise more slowly due to the viscosity increment from the rigid cells than its oxygenated counterpart. When the blood does not sickle, the oxygenated and deoxygenated cells have the same viscosity and rise time. Because the patients own oxygenated blood serves as a control, the method is immune to errors arising from issues such as anemia, polycythemia vera or the presence of malarial parasites. The method is extremely rapid (tens of seconds), robust, and inexpensive. We have developed a simple analytic description of the process, which shows that initially height proceeds as the square root of time, and this is what is observed. The differences in sickle and normal blood are readily observed. Progress in integrating deoxygenation of the blood into the device will also be presented.

Ratchets, red cells, and metastability

Sickle cell disease is a genetic disorder in which a negatively charged glutamic acid is replaced by a hydrophobic valine on the surface of the hemoglobin molecule, leading to polymerization of the deoxygenated form, and resulting in microvascular obstruction. Because of the high volume occupancy under which polymerization occurs physiologically, this process has been an exemplar in the study of excluded volume effects on assembly. More recently, we have identified yet another type of crowding effect involving the obstruction of the ends at which the polymers grow as a consequence of the dense arrays in which these polymers form. This makes such solutions metastable, and leads to Brownian ratchet behavior in which pressure is exerted outward when the gel occupies a finite volume, as in an emulsion or red cell. Such behavior is capable of holding sickled cells in place in the microcirculation against weak pressure differentials (hundreds of Pa), but not against the typical pressures found in vivo.