Interferometric Phase Microscopy Research Articles

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.

Detection of bladder cancer cells using quantitative interferometric label-free imaging flow cytometry.

Bladder cancer is one of the most common cancers with a high recurrence rate. Patients undergo mandatory yearly scrutinies, including cystoscopies, which makes bladder cancer highly distressing and costly. Here, we aim to develop a non-invasive, label-free method for the detection of bladder cancer cells in urine samples, which is based on interferometric imaging flow cytometry. Eight urothelial carcinoma and one normal urothelial cell lines, along with red and white blood cells, imaged quantitatively without staining by an interferometric phase microscopy module while flowing in a microfluidic chip, and classified by two machine-learning algorithms, based on deep-learning semantic segmentation convolutional neural network and extreme gradient boosting. Furthermore, urine samples obtained from bladder-cancer patients and healthy volunteers were imaged, and classified by the system. We achieved accuracy and area under the curve (AUC) of 99% and 97% for the cell lines on both machine-learning algorithms. For the real urine samples, the accuracy and AUC were 96% and 96% for the deep-learning algorithm and 95% and 93% for the gradient-boosting algorithm, respectively. By combining label-free interferometric imaging flow cytometry with high-end classification algorithms, we achieved high-performance differentiation between healthy and malignant cells. The proposed technique has the potential to supplant cystoscopy in the bladder cancer surveillance and diagnosis space.

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.

Quasi-common-path off-axis interferometric quantitative phase microscopy based on amplitude-division

A quasi-common-path off-axis interferometric quantitative phase microscopy (iQPM) is proposed using amplitude-division in this paper. Utilizing a quasi-common-path interferometric module, our method divides the object beam into two copies using a beam-splitter and two mirrors after the output of the microscope, while spatially filters one of the copies to act as the reference beam, and thus yields off-axis interference at the camera plane. The compact module is built using simple optical elements without the requirements of special optical skills and/or complex alignment. In contrast to the previous common-path off-axis iQPMs, the proposed method can flexibly adjust each channel to improve the visibility and/or frequency of the interferogram. Several experimental results are presented to demonstrate the validity and stability of the proposed iQPM.

Real-Time Stain-Free Classification of Cancer Cells and Blood Cells Using Interferometric Phase Microscopy and Machine Learning.

We present a method for real-time visualization and automatic processing for detection and classification of untreated cancer cells in blood during stain-free imaging flow cytometry using digital holographic microscopy and machine learning in throughput of 15 cells per second. As a preliminary model for circulating tumor cells in the blood, following an initial label-free rapid enrichment stage based on the cell size, we applied our holographic imaging approach, providing the quantitative optical thickness profiles of the cells during flow. We automatically classified primary and metastatic colon cancer cells, where the two types of cancer cells were isolated from the same individual, as well as four types of blood cells. We used low-coherence off-axis interferometric phase microscopy and a microfluidic channel to image cells during flow quantitatively. The acquired images were processed and classified based on their morphology and quantitative phase features during the cell flow. We achieved high accuracy of 92.56% for distinguishing between the cells, enabling further automatic enrichment and cancer-cell grading from blood. © 2020 International Society for Advancement of Cytometry.

Label-free discrimination and selection of cancer cells from blood during flow using holography-induced dielectrophoresis.

We present a method for label-free imaging and sorting of cancer cells in blood, which is based on a dielectrophoretic microfluidic chip and label-free interferometric phase microscopy. The chip used for imaging has been embedded with dielectrophoretic electrodes, and therefore it can be used to sort the cells based on the decisions obtained during the cell flow by the label-free quantitative imaging method. Hence, we obtained a real-time, automatic, label-free imaging flow cytometry with the ability to sort the cells during flow. To validate our model, we combined into the label-free imaging interferometer a fluorescence imaging channel that indicated the correctness of the label-free sorting. We have achieved above 98% classification success and 69% sorting accuracy at flow rates of 4 to 7 μL hr-1 . In the future, this method is expected to help in label-free sorting of circulating tumor cells in blood following an initial state-of-the-art cell enrichment.

Cell and nucleus refractive-index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy.

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co-registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.

Stain-free interferometric phase microscopy correlation with DNA fragmentation stain in human spermatozoa.

Acridine orange (AO) staining is used to diagnose DNA fragmentation status in sperm cells. Interferometric phase microscopy (IPM) is an optical imaging method based on digital holographic microscopy that provides quantitative morphological and refractive index imaging of cells in vitro without the need for staining. We have imaged sperm cells using stain-free IPM in order to estimate different cellular parameters, such as acrosome dry mass and size, in addition to an embryologist evaluation according to the World Health Organization (WHO)-2010 criteria. Following this, the same sperm cells were stained by AO, imaged using a fluorescence confocal microscope and assessed by the AO-emitted color, forming five DNA fragmentation groups. These DNA fragmentation groups were correlated with the embryologist-based classification and the IPM-based morphological parameters. Our results indicate on significant differences in the IPM-based parameters between groups with different fragmentation levels. Based on the validation with AO, we conclude that stain-free IPM images analyzed digitally may assist in selecting sperm cells with intact DNA prior to intracytoplasmic sperm injection. This information may potentially increase percentage of successful pregnancies.

Two-exposure quasi-common-path point diffraction interferometric phase microscopy using a four-step algorithm

A two-exposure nearly common-path point diffraction interferometric phase microscopy (IPM) is presented using polarization modulation and one-step grating shifting to implement quantitative phase imaging. The IPM is constructed by an improved Michelson configuration with a reflective grating, and its frequency spectrum generated by a circularly polarized object beam makes double copies through a beam splitter. One copy is low-pass filtered and reflected by a reflective pinhole mirror to create a reference beam, and the other copy is converted by a polarizer and then reflected by a reflective grating to achieve a 45° linearly polarized object beam. By the combination of a polarizing cube beam splitter with 45° tilted angle and a translation of the reflective grating with a π phase shift, four interferograms with π/2 phase shift can be obtained in two exposures. The standard four-step algorithm can then be used to reconstruct the phase of the specimen. The utility of the proposed method was demonstrated with measurements on a phase plate, cells and an oil drop.

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.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry.

Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging.

We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

Quantitative phase microscopy spatial signatures of cancer cells.

We present cytometric classification of live healthy and cancerous cells by using the spatial morphological and textural information found in the label-free quantitative phase images of the cells. We compare both healthy cells to primary tumor cells and primary tumor cells to metastatic cancer cells, where tumor biopsies and normal tissues were isolated from the same individuals. To mimic analysis of liquid biopsies by flow cytometry, the cells were imaged while unattached to the substrate. We used low-coherence off-axis interferometric phase microscopy setup, which allows a single-exposure acquisition mode, and thus is suitable for quantitative imaging of dynamic cells during flow. After acquisition, the optical path delay maps of the cells were extracted and then used to calculate 15 parameters derived from the cellular 3D morphology and texture. Upon analyzing tens of cells in each group, we found high statistical significance in the difference between the groups in most of the parameters calculated, with the same trends for all statistically significant parameters. Furthermore, a specially designed machine learning algorithm, implemented on the phase map extracted features, classified the correct cell type (healthy/cancer/metastatic) with 81-93% sensitivity and 81-99% specificity. The quantitative phase imaging approach for liquid biopsies presented in this paper could be the basis for advanced techniques of staging freshly isolated live cancer cells in imaging flow cytometers. © 2017 International Society for Advancement of Cytometry.

Compact interferometric module for full-field interferometric phase microscopy with low spatial coherence illumination.

We propose a compact and external off-axis interferometric module that can achieve interference with low spatial coherence illumination over the entire field of view. The interferometer is easy to align and stable and can be connected to the output of an existing microscope illuminated with a low spatial coherence light source, thus allowing quantitative phase imaging with a low degree of spatial noise. We demonstrate the imaging and the interference properties of the proposed interferometric module and use it for quantitative phase imaging of reflective samples.

Interferometric phase microscopy using slightly-off-axis reflective point diffraction interferometer

An interferometric phase microscopy (IPM) is proposed using slightly-off-axis reflective point diffraction interferometry for quantitative phase imaging. A retro-reflector consisting two mirrors is used to generate an angle between the object beam and reference beam, and a 45° tilted polarizing beam splitter is used to split the horizontal and vertical components of the both beams. Two carrier interferograms with π/2 phase-shift can be acquired in one shot, and the phase distribution of a thin specimen can be retrieved using a fast reconstruction method. The new IPM without loss in the utilization of the input-plane field of view combines the real time and optimizing detector bandwidth measurement benefit associated with slightly-off-axis method, high stability associated with common path geometry, and simplicity in terms of procedure and setup. Experiments are carried out on both static and dynamic specimens to demonstrate the validity and stability of the proposed method.

Combined optical micromanipulation and interferometric topography (COMMIT).

Optical tweezers have emerged as a prominent light-based tool for pico-Newton (pN) force microscopy in mechanobiological studies. However, the efficacy of optical tweezers are limited in applications where concurrent metrology of the nano-sized structures under interrogation is essential to the quantitative analysis of its mechanical properties and various mechanotransduction events. We have developed an all-optical platform delivering pN force resolution in parallel with nano-scale structural imaging of the biological sample by combining optical tweezers with interferometric quantitative phase microscopy. These capabilities allow real-time micromanipulation and label-free measurement of sample's nanostructures and nanomechanical responses, opening avenues to a wide range of new research possibilities and applications in biology.

Label-Free Quantitative Imaging of Sperm for In-Vitro Fertilization Using Interferometric Phase MicroscopyIVF, IMSI, ICSI, IPM, Holography, Imaging, Sperm

Accurate morphological evaluation via imaging of sperm cells is a critical step in both routine sperm analysis and sperm cell selection for in-vitro fertilization. In this paper, we review the significant advantages of interferometric phase microscopy (IPM), also called digital holographic microscopy, for label-free sperm imaging in fertility clinics. We first review the current state of the art of label-free sperm cell imaging for in-vitro fertilization, including bright-field, Zernike phase contrast, differential interference contrast (DIC), Hoffman modulation contrast and polarized light microscopy, as well as intracytoplasmic morphologically selected sperm injection (IMSI). Using experimental demonstrations, we compare the limitations of these techniques for label-free sperm imaging. IPM and its advantages are then discussed in detail. These include the ability to receive quantitative data regarding the 3D structure of sperm cells, as well as data concerning sperm biomass and morphology without labeling. The current gap to clinical use is discussed, as well as the possible solution of employing compact and simple holographic modules, which may allow holographic imaging to reach the clinic in the near future.

Prediction of photothermal phase signatures from arbitrary plasmonic nanoparticles and experimental verification

We present a new approach for predicting spatial phase signals originating from photothermally excited metallic nanoparticles of arbitrary shapes and sizes. The heat emitted from such a nanoparticle affects the measured optical phase signal via changes in both the refractive index and thickness of the nanoparticle surroundings. Because these particles can be bio-functionalized to bind certain biological cell components, they can be used for biomedical imaging with molecular specificity, as new nanoscopy labels, and for photothermal therapy. Predicting the ideal nanoparticle parameters requires a model that computes the thermal and phase distributions around the particle, thereby enabling more efficient phase imaging of plasmonic nanoparticles and avoiding trial-and-error experiments while using unsuitable nanoparticles. The proposed nonlinear model is the first to enable the prediction of phase signatures from nanoparticles with arbitrary parameters. The model is based on a finite-volume method for geometry discretization and an implicit backward Euler method for solving the transient inhomogeneous heat equation, followed by calculation of the accumulative phase signal. To validate the model, we compared its results with experimental results obtained for gold nanorods of various concentrations, which we acquired using a custom-built wide-field interferometric phase microscopy system. A nonlinear computational model can accurately predict thermally induced phase changes originated from arbitrarily shaped plasmonic nanoparticles. The model, developed by Omry Blum and Natan Shaked of Tel Aviv University in Israel, first numerically calculates the thermal distributions around the photothermally excited nanoparticles, and then generates the associated phase maps for light transmitted through the particle sample. Predictions generated by the model agreed well with both previous thermal-distribution studies and new experimental results for gold nanorods obtained using interferometric phase microscopy. Since plasmonic nanoparticles can be used as either imaging-contrast agents or heat-therapy agents in biomedical applications, using such a model can predict the performance of arbitrary shaped plasmonic nanoparticles even prior to their actual synthesis. Thus, this model can be used to enhance the nanoparticle performance in phase-imaging or heat-therapy techniques.

Interferometric phase microscopy for label-free morphological evaluation of sperm cells

To compare label-free interferometric phase microscopy (IPM) to label-free and label-based bright-field microscopy (BFM) in evaluating sperm cell morphology. This comparison helps in evaluating the potential of IPM for clinical sperm analysis without staining. Comparison of imaging modalities. University laboratory. Sperm samples were obtained from healthy sperm donors. We evaluated 350 sperm cells, using portable IPM and BFM, according to World Health Organization (WHO) criteria. The parameters evaluated were length and width of the sperm head and midpiece; size and width of the acrosome; head, midpiece, and tail configuration; and general normality of the cell. Continuous variables were compared using the Student's t test. Categorical variables were compared with the χ(2) test of independence. Sensitivity and specificity of IPM and label-free BFM were calculated and compared with label-based BFM. No statistical differences were found between IPM and label-based BFM in the WHO criteria. In contrast, IPM measurements of head and midpiece width and acrosome area were different from those of label-free BFM. Sensitivity and specificity of IPM were higher than those of label-free BFM for the WHO criteria. Label-free IPM can identify sperm cell abnormalities, with an excellent correlation with label-based BFM, and with higher accuracy compared with label-free BFM. Further prospective clinical trials are required to enable IPM as part of clinical sperm selection procedures.

Parallel two-step spatial carrier phase-shifting interferometric phase microscopy with fast phase retrieval

A method of parallel two-step spatial carrier phase-shifting two-window interferometer with fast phase retrieval is applied for microscopic imaging. The method prior captures interferograms offline without any sample only once. As a consequence, the interferograms for a phase sample can be acquired by camera in a single shot, and the phase distribution of the sample can be then reconstructed without phase unwrapping process if the phase variance is less than 2π. This method can eliminate the aberration and noise of the system and phase tilt at the same time, and thus improve the phase retrieval speed. Experiments are demonstrated to prove the validation of the presented method.