Label-free, high-throughput holographic imaging to evaluate mammalian gametes and embryos†.

Assisted reproduction is one of the significant tools to treat human infertility. Morphological assessment is the primary method to determine sperm and embryo viability during in vitro fertilization cycles. It has the advantage of being a quick, convenient, and inexpensive means of assessment. However, visual observation is of limited predictive value for early embryo morphology. It has led many to search for other imaging tools to assess the reproductive potential of a given embryo. The limitations of visual assessment apply to both humans and animals. One recent innovation in assisted reproduction technology imaging is interferometric phase microscopy, also known as holographic microscopy. Interferometric phase microscopy/quantitative phase imaging is the next likely progression of analytical microscopes for the assisted reproduction laboratory. The interferometric phase microscopy system analyzes waves produced by the light as it passes through the specimen observed. The microscope collects the light waves produced and uses the algorithm to create a hologram of the specimen. Recently, interferometric phase microscopy has been combined with quantitative phase imaging, which joins phase contrast microscopy with holographic microscopy. These microscopes collect light waves produced and use the algorithm to create a hologram of the specimen. Unlike other systems, interferometric phase microscopy can provide a quantitative digital image, and it can make 2D and 3D images of the samples. This review summarizes some newer and more promising quantitative phase imaging microscopy systems for evaluating gametes and embryos. Studies clearly show that quantitative phase imaging is superior to bright field microscopy-based evaluation methods when evaluating sperm and oocytes prior to IVF and embryos prior to transfer. However, further assessment of these systems for efficacy, reproducibility, cost-effectiveness, and embryo/gamete safety must take place before they are widely adopted.

Analyzing Blood Cells of High-Risk Myelodysplastic Syndrome Patients Using Interferometric Phase Microscopy and Fluorescent Flow Cytometry.

Myelodysplastic syndromes (MDSs) are a group of potentially deadly diseases that affect the morphology and function of neutrophils. Rapid diagnosis of MDS is crucial for the initiation of treatment that can vastly improve disease outcome. In this work, we present a new approach for detecting morphological differences between neutrophils isolated from blood samples of high-risk MDS patients and blood bank donors (BBDs). Using fluorescent flow cytometry, neutrophils were stained with 2',7'-dichlorofluorescin diacetate (DCF), which reacts with reactive oxygen species (ROS), and Hoechst, which binds to DNA. We observed that BBDs possessed two cell clusters (designated H and L), whereas MDS patients possessed a single cluster (L). Later, we used FACS to sort the H and the L cells and used interferometric phase microscopy (IPM) to image the cells without utilizing cell staining. IPM images showed that H cells are characterized by low optical path delay (OPD) in the nucleus relative to the cytoplasm, especially in cell vesicles containing ROS, whereas L cells are characterized by low OPD in the cytoplasm relative to the nucleus and no ROS-containing vesicles. Moreover, L cells present a higher average OPD and dry mass compared to H cells. When examining neutrophils from MDS patients and BBDs by IPM during flow, we identified ~20% of cells as H cells in BBDs in contrast to ~4% in MDS patients. These results indicate that IPM can be utilized for the diagnosis of complex hematological pathologies such as MDS.

Individual sperm selection by microfluidics integrated with interferometric phase microscopy.

The selection of sperm cells possessing normal morphology and motility is crucial for many assisted reproductive technologies (ART), especially for intracytoplasmic sperm injection (ICSI), as sperm quality directly affects the probability of inducing healthy pregnancy. We present a novel platform for real-time quantitative analysis and selection of individual sperm cells without staining. Towards this end, we developed an integrated approach, combining interferometric phase microscopy (IPM), for stain-free sperm imaging and real-time automatic analysis based on the sperm cell 3D morphology and contents, with a disposable microfluidic device, for sperm selection and enrichment. On testing the capabilities of the microfluidic device, we obtained successful selection of sperm cells with a selectivity of 89.5±3.5%, with no negative-decision sperm cells being inadvertently selected. In addition, we demonstrate the accuracy of sperm cell analysis using IPM by comparing the quantitative analysis produced by our IPM-based algorithm to the qualitative visual analysis performed independently by an experienced embryologist, which resulted in precision and specificity of 100%. We believe that the presented integrated approach has the potential to dramatically change the way sperm cells are selected for ICSI and other ART procedures, making the selection process more objective, quantitative and automatic, and thereby increasing success rates.

Localized measurements of physical parameters within human sperm cells obtained with wide-field interferometry.

We developed a new method to identify the separate cellular compartments in the optical path delay (OPD) maps of un-labeled spermatozoa. This was conducted by comparing OPD maps of fixed, un-labeled spermatozoa to bright field images of the same cells following labeling. The labeling enabled us to identify the acrosomal and nuclear compartments in the corresponding OPD maps of the cells. We then extracted the refractive index maps of fixed cells by dividing the OPD maps of spermatozoa by the corresponding thickness maps of the same cells, obtained with AFM. Finally, the dry mass of the head, nucleus and acrosome of un-labeled immobile spermatozoa, was measured. This method provides the ability to quantitatively measure the dry mass of cellular compartments within human spermatozoa. We expect that these measurements will assist label-free selection of sperm cells for fertilization.