High-resolution transport-of-intensity quantitative phase microscopy with annular illumination

For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively addressed by replacing the conventional circular illumination aperture with an annular one. The matched annular illumination not only strongly boosts the phase contrast for low spatial frequencies, but significantly improves the practical imaging resolution to near the incoherent diffraction limit. By incorporating high-numerical aperture (NA) illumination as well as high-NA objective, it is shown, for the first time, that TIE phase imaging can achieve a transverse resolution up to 208 nm, corresponding to an effective NA of 2.66. Time-lapse imaging of in vitro Hela cells revealing cellular morphology and subcellular dynamics during cells mitosis and apoptosis is exemplified. Given its capability for high-resolution QPI as well as the compatibility with widely available brightfield microscopy hardware, the proposed approach is expected to be adopted by the wider biology and medicine community.Comment: This manuscript was originally submitted on 20 Feb. 201

Orientation-independent differential interference contrast microscopy

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Applied Optics 45 (2006): 460-469, doi:10.1364/AO.45.000460.The image in a regular DIC microscope reflects the orientation of the prism shear direction and the optical path gradients in a phase specimen. If the shear direction lies parallel to the specimen boundary no contrast is generated. Also a bias retardance is generally introduced, which creates a gray background and reduces image contrast. Here we describe the theoretical foundation for a new DIC technique, which records phase gradients independently of their orientation and with the digitally generated gradient magnitude image as well as the optical path distribution image free from the gray background. Separate images can show the magnitude distribution of the optical path gradients and of the azimuths, or the two images can be combined into one picture e.g., with the brightness representing magnitudes and color showing azimuths respectively. For experimental verification of the proposed technique we investigated various specimens such as glass rods embedded in Permount, Siemens star nano-fabricated in 90-nm thick silicon oxide layer, Bovine pulmonary artery endothelial cell, etc, using regular DIC optics on a microscope equipped with a precision rotating stage. Several images were recorded with the specimen oriented in different directions, but with the prism bias unchanged, followed by digital alignment and processing of the images. The results demonstrate that the proposed DIC technique can successfully image and measure phase gradients of transparent specimens, independent of the directions of the gradient. The orientation-independent DIC data obtained can also be used to compute the quantitative distribution of specimen phase or to generate enhanced, regular DIC images with any desired shear direction. We are currently developing a new device using special DIC prisms, which allows the bias and shear directions to be switched rapidly without the need to mechanically rotate the specimen or the prism (US Patent Application 2005-0152030). With the new system an orientation independent DIC image should be obtained in a fraction of a second. A detailed description of the new system will be given in a future publication

Quantitative orientation-independent differential interference contrast microscope with fast switching shear direction and bias modulation

Author Posting. © Optical Society of America, 2013. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Optical Society of America A: Optics, Image Science, and Vision 30 (2013): 769-782, doi:10.1364/JOSAA.30.000769.We describe a quantitative orientation-independent differential interference contrast (DIC) microscope, which allows bias retardation to be modulated and shear directions to be switched rapidly without any mechanical movement. The shear direction is switched by a regular liquid-crystal cell sandwiched between two standard DIC prisms. Another liquid-crystal cell modulates the bias. Techniques for measuring parameters of DIC prisms and calibrating the bias are shown. Two sets of raw DIC images with the orthogonal shear directions are captured within 1 s. Then the quantitative image of optical path gradient distribution within a thin optical section is computed. The gradient data are used to obtain a quantitative distribution of the optical path, which represents the refractive index gradient or height distribution. Computing enhanced regular DIC images with any desired shear direction is also possible.This publication was made possible by Grant No. R01-GM101701 from the National Institute of General Medical Sciences, National Institutes of Health

Lensfree computational microscopy tools for cell and tissue imaging at the point-of-care and in low-resource settings.

The recent revolution in digital technologies and information processing methods present important opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their relative cost and complexity. Lensfree computational microscopy is rapidly emerging toward this end, and by discarding lenses and other bulky optical components of conventional imaging systems, and relying on digital computation instead, it can achieve both reflection and transmission mode microscopy over a large field-of-view within compact, cost-effective and mechanically robust architectures. Such high throughput and miniaturized imaging devices can provide a complementary toolset for telemedicine applications and point-of-care diagnostics by facilitating complex and critical tasks such as cytometry and microscopic analysis of e.g., blood smears, Pap tests and tissue samples. In this article, the basics of these lensfree microscopy modalities will be reviewed, and their clinically relevant applications will be discussed

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. © 2012 Jin et al

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

<p>Abstract</p> <p>Background</p> <p>Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions.</p> <p>Results</p> <p>We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells.</p> <p>Conclusion</p> <p>We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.</p