3-D refractive index (RI) distribution is an intrinsic bio-marker for the chemical and structural information about biological cells. Here we develop an optical diffraction tomography technique for the real-time reconstruction of 3-D RI distribution, employing sparse angle illumination and a graphic processing unit (GPU) implementation. The execution time for the tomographic reconstruction is 0.21 s for 96(3) voxels, which is 17 times faster than that of a conventional approach. We demonstrated the real-time visualization capability with imaging the dynamics of Brownian motion of an anisotropic colloidal dimer and the dynamic shape change in a red blood cell upon shear flow.

Abstract:The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs. References and links1. L. Balagopalan, E. Sherman, V. A. Barr, and L. E. Samelson, "Imaging techniques for assaying lymphocyte activation in action," Nat. Rev. Immunol. 11(1), 21-33 (2011). 2. J. C. Edwards and G. Cambridge, "B-cell targeting in rheumatoid arthritis and other autoimmune diseases," Nat.Rev. Immunol. 6(5), 394-403 (2006). 3. M. T. Heneka, M. P. Kummer, and E. Latz, "Innate immune activation in neurodegenerative disease," Nat. Rev.Immunol. 14(7), 463-477 (2014). 4. W. Zou and N. P. Restifo, "T(H)17 cells in tumour immunity and immunotherapy," Nat. Rev. Immunol. 10(4), 248-256 (2010). 41. K. E. Handwerger, J. A. Cordero, and J. G. Gall, "Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure," Mol.

We present an optical holographic micro-tomographic technique for imaging both the three-dimensional structures and dynamics of biological cells. Optical light field images of a sample, illuminated by a plane wave with various illumination angles, are measured in a common-path interferometry, and thus both the three-dimensional refractive index tomogram and two-dimensional dynamics of live biological cells are measured with extremely high sensitivity. The applicability of the technique is demonstrated through quantitative and measurements of morphological, chemical, and mechanical parameters at the individual cell level.

Two-dimensional angle-resolved light scattering maps of individual rod-shaped bacteria are measured at the single-cell level. Using quantitative phase imaging and Fourier transform light scattering techniques, the light scattering patterns of individual bacteria in four rod-shaped species (Bacillus subtilis, Lactobacillus casei, Synechococcus elongatus, and Escherichia coli) are measured with unprecedented sensitivity in a broad angular range from −70° to 70°. The measured light scattering patterns are analyzed along the two principal axes of rod-shaped bacteria in order to systematically investigate the species-specific characteristics of anisotropic light scattering. In addition, the cellular dry mass of individual bacteria is calculated and used to demonstrate that the cell-to-cell variations in light scattering within bacterial species is related to the cellular dry mass and growth.

Rapid identification of bacterial species is crucial in medicine and food hygiene. In order to achieve rapid and label-free identification of bacterial species at the single bacterium level, we propose and experimentally demonstrate an optical method based on Fourier transform light scattering (FTLS) measurements and statistical classification. For individual rod-shaped bacteria belonging to four bacterial species (Listeria monocytogenes, Escherichia coli, Lactobacillus casei, and Bacillus subtilis), two-dimensional angle-resolved light scattering maps are precisely measured using FTLS technique. The scattering maps are then systematically analyzed, employing statistical classification in order to extract the unique fingerprint patterns for each species, so that a new unidentified bacterium can be identified by a single light scattering measurement. The single-bacterial and label-free nature of our method suggests wide applicability for rapid point-of-care bacterial diagnosis.

Optical measurements of the morphological and biochemical imaging of phytoplankton are presented. Employing quantitative phase imaging techniques, 3-D refractive index maps and high-resolution 2-D quantitative phase images of individual live phytoplankton are simultaneously obtained without exogenous labeling agents. In addition, biochemical information of individual phytoplankton including volume, mass, and density of individual phytoplankton are also quantitatively obtained from the measured refractive index distributions. We expect the present method to become a powerful tool for the study of phytoplankton.

Here, the actions of melittin, the active molecule of apitoxin or bee venom, were investigated on human red blood cells (RBCs) using quantitative phase imaging techniques. High-resolution real-time 3-D refractive index (RI) measurements and dynamic 2-D phase images of individual melittin-bound RBCs enabled in-depth examination of melittin-induced biophysical alterations of the cells. From the measurements, morphological, intracellular, and mechanical alterations of the RBCs were analyzed quantitatively. Furthermore, leakage of haemoglobin (Hb) inside the RBCs at high melittin concentration was also investigated.

Background: Diabetic effects on blood rheology are important topics and have been extensively investigated. In particular, it has been known that diabetic RBCs exhibit significantly deteriorated deformability compared to healthy RBCs. Despite the importance of previously measured mechanical properties of diabetic RBCs, several problems remain to be elucidated. First, conventional techniques have difficulties in measuring intrinsic properties of individual cells. Detailed quantification of diabetic alteration in RBCs at the individual cell level is also hard to achieve. Accordingly, previous approaches do not allow the systematical correlative analyses to be performed on simultaneously measured various cell parameters. As a consequence, there are still no general agreements on the integrated effects of diabetic complications or hyperglycemia on individual human RBCs. Methods: To effectively settle the arisen problems, we quantitatively and non-invasively measured the individual RBCs from diabetic and healthy blood donors to figure out the characteristics of diabetic RBCs by employing the quantitative phase imaging (QPI) technique. From the reconstructed three-dimensional (3-D) refractive index (RI) tomogram and the time-series of 2-D phase map of individual RBCs, the morphological (volume, surface area, and sphericity), biochemical (Hb concentration and Hb content), and mechanical (membrane fluctuation) parameters were retrieved. Systematic correlative analyses among measured six RBC parameters and independently determined HbA1c level, defined as the ratio of HbA1c to total Hb, were also performed. In measuring red cells, only gently sedimented RBCs of discocytes were selected. After excluding erroneously measured RBCs, 40, 40, 40, 38, 39, 40 control RBCs of six healthy donors and 40, 40, 40, 40, 40, 40 diabetic RBCs of six diabetic patients were finally analyzed respectively for the study of diabetic effects on characteristics of individual human RBCs. Results: HbA1c level is defined as a percentage of HbA1c, the majority of glycated Hbs in RBC, to total Hb contents in blood. Because HbA1c is formed through 120-days long, non-enzymatic glycation process of Hb, HbA1c level has been perceived as an effective mean to estimate mean glucose concentration of individuals for the last three months. In other words, HbA1c reflects the degree of hyperglycemia. The most noticeable alterations in diabetic RBCs can be found when mean RBC membrane fluctuations of individuals are rearranged in the increasing order of the measured HbA1c levels. The mean values of membrane fluctuations for RBCs are 59.7 ¡¾ 6.2, 58.3 ¡¾ 4.9, 55.0 ¡¾ 5.4, 57.1 ¡¾ 7.4, 51.0 ¡¾ 6.8, and 49.6 ¡¾ 4.6 nm for healthy volunteers, and 45.1 ¡¾ 4.0, 46.9 ¡¾ 3.4, 48.8 ¡¾ 4.0, 47.6 ¡¾ 4.2, 43.0 ¡¾ 4.1, and 48.0 ¡¾ 5.0 nm for diabetic patients, in order of increasing HbA1c. As shown in Fig. 4, mean membrane fluctuation of non-diabetic RBCs tends to decrease as the HbA1c level increases (Pearson correlation coefficient of -0.87 with a p-value of 0.02). Beyond this level of HbA1c, RBCs from diabetic patients exhibit significantly decreased and settled membrane fluctuation. This observed negative correlation between RBC membrane fluctuation and HbA1 is also conceptually in accordance with the report of the previous study. Conclusions: We quantitatively and non-invasively characterize the morphological, biochemical, and mechanical alterations of individual RBCs by diabetes mellitus. HbA1c levels of subjects, which reflect one's mean blood glucose concentration of last three months, were also determined using hematology analyzers. Accordingly, correlative analyses among the retrieved individual RBC parameters and HbA1c level were performed to search unique features which cannot be addressed by conventional techniques with incapability of simultaneous measurement of those parameters. Figure Box plot of membrane fluctuation for measured RBCs from six healthy controls and diabetic patients in increasing order of HbA1c. Boxes, median values with upper and lower quartiles. Short horizontal lines and error bars in each box plot denote the mean values and standard deviations, respectively. Figure. Box plot of membrane fluctuation for measured RBCs from six healthy controls and diabetic patients in increasing order of HbA1c. Boxes, median values with upper and lower quartiles. Short horizontal lines and error bars in each box plot denote the mean values and standard deviations, respectively. Disclosures No relevant conflicts of interest to declare.