High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension.

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine.

Single-cell measurement of red blood cell oxygen affinity.

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

A Microfluidic Approach for Inducing Cell Rotation by Means of Hydrodynamic Forces.

Microfluidic technology allows to realize devices in which cells can be imaged in their three-dimensional shape. However, there are still some limitations in the method, due to the fact that cells follow a straight path while they are flowing in a channel. This can result in a loss in information, since only one side of the cell will be visible. Our work has started from the consideration that if a cell rotates, it is possible to overcome this problem. Several approaches have been proposed for cell manipulation in microfluidics. In our approach, cells are controlled by only taking advantages of hydrodynamic forces. Two different devices have been designed, realized, and tested. The first device induces cell rotation in a plane that is parallel (in-plane) to the observation plane, while the second one induce rotation in a plane perpendicular (out-of-plane) to the observation plane.

Quantitative absorption cytometry for measuring red blood cell hemoglobin mass and volume.

We present an optical system, called the quantitative absorption cytometer (QAC), to measure the volume and hemoglobin mass of red blood cells flowing through a microfluidic channel. In contrast to clinical hematology analyzers, where cells are sphered in order for both volume and hemoglobin to be measured accurately, the QAC measures cells in their normal physiological shape. Human red blood cells are suspended in a refractive index-matching absorbing buffer, driven through a microfluidic channel, and imaged using a transmission light microscope onto a color camera. A red and a blue LED illuminate cells and images at each color are used to independently retrieve cell volume and hemoglobin mass. This system shows good agreement with red blood cell indices retrieved by a clinical hematology analyzer and in fact measures a smaller coefficient of variation of hemoglobin concentration. In addition to cell indices, the QAC returns height and mass maps of each measured cell. These quantitative images are valuable for analyzing the detailed morphology of individual cells as well as statistical outliers found in the data. We also measured red blood cells in hypertonic and hypotonic buffers to quantify the correlation between volume and hemoglobin mass under osmotic stress. Because this method is invariant to cell shape, even extremely nonspherical cells in hypertonic buffers can be measured accurately.

Polarization encoded color camera.

Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared.

Optical determination of intracellular water in apoptotic cells.

Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport-of-intensity equation. At the same time, cell thickness was determined by transmission through an externally applied dye ('transmission-through-dye' microscopy); switching between these two imaging modalities was accomplished by simply changing the illumination wavelength. The sets of data thus obtained allow computation of the refractive index and cell water content within individual cells. The method was illustrated using cells treated with apoptotic agents staurosporine and actinomycin D and with necrosis inducer ionomycin. Water imaging allows discrimination between apoptotic volume decrease due to dehydration from that due to detachment of apoptotic bodies and can be used on samples where cell volume determination alone would be difficult or insufficient.

Fluorescence imaging of flowing cells using a temporally coded excitation.

Imaging fluorescence in moving cells is fundamentally challenging because the exposure time is constrained by motion-blur, which limits the available signal. We report a method to image fluorescently labeled leukemia cells in fluid flow that has an effective exposure time of up to 50 times the motion-blur limit. Flowing cells are illuminated with a pseudo-random excitation pulse sequence, resulting in a motion-blur that can be computationally removed to produce near diffraction-limited images. This method enables observation of cellular organelles and their behavior in a fluid environment that resembles the vasculature.

Dye exclusion microfluidic microscopy.

We present an optical system to measure height maps of non-adherent cells as they flow through a microfluidic channel. The cells are suspended in an index-matching absorbing buffer, where cell height is evaluated by measuring the difference in absorption between the cell and the background. Unlike interferometric microscopes, the measured cell height is nearly independent of the cell's optical properties. The height maps are captured using a single exposure of a color camera, and consequently the system is capable of high-throughput characterization of large collections of cells. Using this system, we have measured more than 1600 height maps and volumes of three different leukemia cell lines.

Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects.

Measuring the pressures across microfluidic droplets with an optical tweezer.

We introduce a novel technique that enables pressure measurements to be made in microfluidic chips using optical trapping. Pressure differentials across droplets in a microfluidic channel are determined by monitoring the displacements of a bead in an optical trap. We provide physical interpretation of the results. Our experiments reveal that our device has high sensitivity and can be operated over a wide range of pressures from several Pascals to several thousand Pascals.

Phase imaging flow cytometry using a focus-stack collecting microscope.

This letter introduces a fluidics-based focus-stack collecting microscope. A microfluidic device transports cells through the focal plane of a microscope, resulting in an efficient method to collect focus stacks of large collections of single cells. Images from the focus stacks are used to reconstruct the quantitative phase of cells with the transport-of-intensity-equation method. Using the phase imaging flow cytometer, we measure three-dimensional shape variations of red blood and leukemia cells.

Reconfigurable imaging systems using elliptical nanowires.

Materials that have subwavelength structure can add degrees of freedom to optical system design that are not possible with bulk materials. We demonstrate two lenses that are composed out of lithographically patterned arrays of elliptical cross-section silicon nanowires, which can dynamically reconfigure their imaging properties in response to the polarization of the illumination. In each element, two different focusing functions are polarization encoded into a single lens. The first nanowire lens has a different focal length for each linear polarization state, thereby realizing the front end of a nonmechanical zoom imaging system. The second nanowire lens has a different optical axis for each linear polarization state, demonstrating stereoscopic image capture from a single physical aperture.

Multicolored vertical silicon nanowires.

We demonstrate that vertical silicon nanowires take on a surprising variety of colors covering the entire visible spectrum, in marked contrast to the gray color of bulk silicon. This effect is readily observable by bright-field microscopy, or even to the naked eye. The reflection spectra of the nanowires each show a dip whose position depends on the nanowire radii. We compare the experimental data to the results of finite difference time domain simulations to elucidate the physical mechanisms behind the phenomena we observe. The nanowires are fabricated as arrays, but the vivid colors arise not from scattering or diffractive effects of the array, but from the guided mode properties of the individual nanowires. Each nanowire can thus define its own color, allowing for complex spatial patterning. We anticipate that the color filter effect we demonstrate could be employed in nanoscale image sensor devices.

A microfluidic fluorescence measurement system using an astigmatic diffractive microlens array.

We demonstrate an opto-fluidic detection system based on an array of astigmatic diffractive microlenses integrated into a microfluidic flow focus device. Each astigmatic microlens produces a line excitation across the channel and collects fluorescence emission from the linear detection regions. The linear excitation spot results in uniform excitation across the channel and high time resolution in the direction of the flow. Collected fluorescence from each integrated microlens is relayed to a sub-region on a fast CMOS camera. By analyzing the signal from individual microlenses, we demonstrate counting and resolution of 500 nm and 1.1 µm beads at rates of up to 8,300 per second at multiple locations. In addition, a cross-correlation analysis of the signals from different microlenses yields the velocity dispersion of beads traveling through the channel at peak speeds as high as 560 mm/s. Arrays of specifically designed diffractive optics promise to increase the resolution and functionality of opto-fluidic analysis such as flow cytometry and fluorescence cross-correlation spectroscopy.

Optical manipulation with planar silicon microring resonators.

We demonstrate optically trapping of microparticles on silicon microring resonators. Once trapped on a microring, a particle can be confined in an optical potential with a depth of 25 k(B)T over the entire microring's circumference. The particles are propelled around the microring at hundreds of micrometers per second, producing periodic revolutions at a few hertz. We anticipate that the increased force and highly accurate positioning obtainable with this system will lead to various nanomanipulation applications.

Scannable plasmonic trapping using a gold stripe.

Using counterpropagating surface plasmon polaritons (SPPs) on a gold stripe, we demonstrate a scannable integrated optical tweezer. We demonstrate the trapping of individual fluorescent beads on the stripe, which supports a single quasi-transverse magnetic (TM) mode at the metal-water interface. The beads are localized to the stripe center, with a standard deviation of 51 nm transverse to the stripe, corresponding to a trap stiffness of 1.7 pN/microm. The localization along the stripe is achieved by balancing the scattering forces from the two counterpropagating SPPs excited by prism coupling. The particle position along the stripe can be controlled by varying the relative intensity of the two input beams. This work adds an important new capability to plasmonic optical tweezers, that of scanning. We anticipate that this will broaden the range of applications of plasmonic optical manipulation.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and ßS-globin. Kinetic studies with MetAP2 show that ßS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood-derived erythroid progenitor 2 cells shows more extensive modification of α-globin than ß-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on ßS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor-treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2-affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

High-throughput fluorescence detection using an integrated zone-plate array.

Microfluidic devices enable massive parallelization of sample manipulation and delivery, but a similarly parallelized and integrated optical detection system does not yet exist. Standard large numerical aperture wide field or scanning optical systems are not capable of the large field of view and detection sensitivity required to collect fluorescence from parallel arrays of microfluidic devices. Instead, we present a fluorescence measurement platform based on a microfabricated zone-plate array integrated into a parallelized microfluidic device. The zone-plate array is orientated so that a single high numerical aperture zone plate is aligned to read out the fluorescence from each of 64 output channels of a drop-making device. The parallelization of microfluidics and optics produces an integrated system capable of analysis of nearly 200,000 drops per second.

Propulsion of gold nanoparticles with surface plasmon polaritons: evidence of enhanced optical force from near-field coupling between gold particle and gold film.

We experimentally demonstrate the enhanced propulsion of gold nanoparticles by surface plasmon polaritons (SPPs). Three dimensional finite difference time domain (FDTD) simulations indicate considerably enhanced optical forces due to the field enhancement provided by SPPs and the near-field coupling between the gold particles and the film. This coupling is an important part of the enhanced propulsion phenomenon. Finally, the measured optical force is compared with that predicted by FDTD simulations and proven to be reasonable.

Total internal reflection photonic crystal prism.

An integrated total internal reflection prism is demonstrated that generates a transversely localized evanescent wave along the boundary between a photonic crystal and an etched out trench. The reflection can be described by either the odd symmetry of the Bloch wave or a tangential momentum matching condition. In addition, the Bloch wave propagates through the photonic crystal in a negative refraction regime, which manages diffraction within the prism. A device with three input channels has been fabricated and tested that illuminates different regions of the reflection interface. The reflected wave is then sampled by a photonic wire array, where the individual channels are resolved. Heterodyne near field scanning optical microscopy is used to characterize the spatial phase variation of the evanescent wave and its decay constant.