Dye exclusion microfluidic microscopy

We present an optical system to measure height maps of nonadherent 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

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

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 (NCT 03282656) 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 NCT 03282656, 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

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 transportof-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 non-mechanical 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 apertureclose91

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

Scanning microscopy using a short-focal-length Fresnel zone plate

We demonstrate a form of scanning microscopy using a short-focal-length Fresnel zone plate and a low-NA relay telescope. Owing to a focal length of only 5 μm, the zone plate produces large wavefront tilt and consequently severe vignetting for off-axis illumination. By scanning an optically trapped fluorescent sphere, we measure the vignetted collection region of the zone-plate imaging system. The fluorescence collection efficiency is sharply peaked and has a lateral width of 550 nm, which agrees with numerical simulations

Visualization 1: Gigapixel multispectral microscopy

Complete spectral stack. Originally published in Optica on 20 July 2015 (optica-2-7-654