Quantitative Differential Interference Contrast Research Articles

Single-exposure quantitative differential interference contrast microscopy using bandlimited image and its Fourier transform constraints

Phase microscopy that records the bandlimited image and its Fourier image simultaneously (BIFT) is a phase retrieval method with unique and rapid convergence. In this paper, we present a single-exposure quantitative differential interference contrast (DIC) microscopy based on BIFT method. The contrasts of the recorded DIC image and its Fourier image, analyzed by simulation and experiment, can be largely improved by the initial phase difference between two sheared lights (bias), however their trends with biases are opposite. By adding the optimized bias with the compromise of the contrasts in image and Fourier space, the phase sensitivity can be improved than BIFT method only. We have experimentally demonstrated that a sample of 25 nm height can be successfully recovered from a single exposure. The presented single-exposure quantitative DIC microscopy provides a promising technique for real-time phase imaging.

Optical absorption and dichroism of single melanin nanoparticles.

Melanin nanoparticles (NPs) have important biological functions including photoprotection and colouration, and artificial melanin-like NPs are relevant for catalysis, drug delivery, diagnosis and therapy. Despite their importance, the optical properties of single melanin NPs have not been measured. We combine quantitative differential interference contrast (qDIC) and extinction microscopy to characterise the optical properties of single NPs, both naturally sourced from cuttlefish ink, as well as synthetic NPs using polydopamine (PDA) and L-3,4-dihydroxyphenylalanine (L-DOPA). Combining qDIC with extinction, we determine the absorption index of individual NPs. We find that on average the natural melanin NPs have a higher absorption index than the artificial melanin NPs. From the analysis of the polarisation-dependent NP extinction, the NP aspect ratio is determined, with mean values at 405 nm wavelength in agreement with transmission electron microscopy. At longer wavelengths, we observe an additional optical anisotropy which is attributed to dichroism by structural ordering of the melanin. Our quantitative analysis yields a dichroism of 2-10% of the absorption index, increasing with wavelength from 455 nm to 660 nm for L-DOPA and PDA. Such an in-depth quantification of the optical properties of single melanin NPs is important for the design and future application of these ubiquitous bionanomaterials.

Inspired By Shinya Inoue: Quantitative Orientation-Independent Differential Interference Contrast Microscopy

Inspired By Shinya Inoue: Quantitative Orientation-Independent Differential Interference Contrast Microscopy

Algorithm based on the 1-norm character of interferograms for quantitative differential interference imaging

Differential interference contrast (DIC) microscopy is widely used as a high-contrast imaging method for observing transparent biological samples. However, the absence of fringes in DIC images results in phase gradients that cannot be effectively extracted by most phase-shift algorithms, limiting its application in quantitative detection. Therefore, we propose an algorithm based on the 1-norm character of interferograms that can accurately extract phase shifts from a series of interferograms with less than 1/2 fringes. We improved this algorithm by combining the calculated phase shift with the least-squares fitting method to further extract the phase and applied it to quantitative DIC imaging. In contrast to the conventional algorithm, this algorithm can extract the phase gradient in DIC images with unknown phase shifts, thereby eliminating the computational error in the conventional algorithm introduced by imprecise phase shifts. Simulations and experiments demonstrated that the proposed algorithm can perform accurate phase gradient extraction.

Sizing individual dielectric nanoparticles with quantitative differential interference contrast microscopy.

We report a method to measure the size of single dielectric nanoparticles with high accuracy and precision using quantitative differential interference contrast (DIC) microscopy. Dielectric nanoparticles are detected optically by the conversion of the optical phase change into an intensity change using DIC. Phase images of individual nanoparticles were retrieved from DIC by Wiener filtering, and a quantitative methodology to extract nanoparticle sizes was developed. Using polystyrene beads of 100 nm radius as size standard, we show that the method determines this radius within a few nm accuracy. The smallest detectable polystyrene bead is limited by background and shot-noise, which depend on acquisition and analysis parameters, including the objective numerical aperture, the DIC phase offset, and the refractive index contrast between particles and their surrounding. Measurements on small beads of 15 nm nominal radius are shown, and a sensitivity limit potentially reaching down to 1.8 nm radius was inferred. As application example, individual nanodiamonds with nominal sizes below 50 nm were measured, and were found to have a nearly exponential size distribution with 28 nm mean value. Considering the importance of dielectric nanoparticles in many fields, from naturally occurring virions to polluting nanoplastics, the proposed method could offer a powerful quantitative tool for nanoparticle analysis, combining accuracy, sensitivity and high-throughput with widely available and easy-to-use DIC microscopy.

Simultaneous microscopic imaging of thickness and refractive index of thin layers by heterodyne interferometric reflectometry (HiRef)

The detection of spatial or temporal variations in very thin samples has important applications in the biological sciences. For example, cellular membranes exhibit changes in lipid composition and order, which in turn modulate their function in space and time. Simultaneous measurement of thickness and refractive index would be one way to observe these variations, yet doing it noninvasively remains an elusive goal. Here we present a microscopic-imaging technique to simultaneously measure the thickness and refractive index of thin layers in a spatially resolved manner using reflectometry. A coherent laser beam, focussed by a high-numerical-aperture microscope objective and reflected by the sample, is measured using its heterodyne interference with a reference in order to determine the amplitude and phase of the reflected field and thus the complex reflection coefficient. Comparing the results with the theoretically calculated reflection of a thin layer under coherent illumination of high numerical aperture by the microscope objective, the refractive index and thickness of the layer are determined. We present results on a layer of polyvinylacetate with a thickness of approximately 80 nm. These results have a precision better than 10% in the thickness and better than 1% in the refractive index. The measured refractive index is consistent with literature values, and the measured thickness is close to measurements of the same sample by quantitative differential interference contrast. We discuss the significance of these results and the possibility of performing accurate measurements on nanometric layers. Notably, the shot-noise limit of the technique is below 0.5 nm in thickness and 0.0005 in refractive index for millisecond measurement times.

Self-compensating quantitative differential interference contrast microscopy imaging system using liquid crystal shearing assembly

A quantitative differential interference contrast microscopy (DICM) using a liquid crystal shearing assembly (LCSA) is proposed and demonstrated experimentally. Differential interference contrast (DIC) imaging with arbitrary shearing amount and different shearing direction can be implemented by changing the driving voltage applied to the liquid crystal without mechanical devices. Moreover, by combining a quarter-wave plate and a mirror, not only the shearing amount is enlarged, but also the aberration and the additional phase shift induced by the refractive index difference between ordinary and extraordinary light can be compensated. Using the proposed LCSA/DICM system, we implement DIC imaging of a polystyrene microsphere crown and the quantitative differential phase measurement of an oral epithelial cell.

RETRACTED: Study of strain-driven martensitic phase transformation rate via data-driven approach

Abstract In the kinetic studies of martensitic phase transformation, the propagation velocity of the phase boundary is considered as the speed of transformation. But there is no interface at the beginning of phase transformation, we cannot track the development of the interface. Here we propose a new mathematical model that describes the rate of martensitic transformation by the data-driven method, which is based on the study of the change in area fraction in each phase. In this paper, the K-means clustering method is used to classify martensite and austenite through advanced quantitative differential interference contrast microscopy system. After classification, the clustering results are in good agreement with microscopic observations. The phase transformation rate for a set of tensile tests under various strain rates is determined. Our work provides a useful reference for modeling the strain-driven martensitic transformation rate.

Lipid Bilayer Thickness Measured by Quantitative DIC Reveals Phase Transitions and Effects of Substrate Hydrophilicity.

Quantitative differential interference contrast microscopy is demonstrated here as a label-free method, which is able to image and measure the thickness of lipid bilayers with 0.1 nm precision. We investigate the influence of the substrate on the thickness of fluid-phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-supported lipid bilayers and find a thinning of up to 10%, depending on substrate hydrophilicity, local bilayer coverage, and ionic strength of the medium. With fluorescently labeled lipid bilayers, we also observe changes in the bilayer thickness depending on the choice of fluorophore. Furthermore, liquid-ordered domains in bilayers, formed from DOPC, cholesterol, and sphingomyelin, are measured, and the corresponding thickness change between the liquid-ordered and liquid-disordered phases is accurately determined. Again, the thickness difference is found to be dependent on the presence of the fluorophore label, highlighting the need for quantitative label-free techniques.

Lung cancer diagnosis with quantitative DIC microscopy and a deep convolutional neural network.

We present a study on lung squamous cell carcinoma diagnosis using quantitative TI-DIC microscopy and a deep convolutional neural network (DCNN). The 2-D phase map of unstained tissue sections is first retrieved from through-focus differential interference contrast (DIC) images based on the transport of intensity equation (TIE). The spatially resolved optical properties are then computed from the 2-D phase map via the scattering-phase theorem. The scattering coefficient ( ) and the reduced scattering coefficient ( ) are found to increase whereas the anisotropy factor (g) is found to decrease with cancer. A DCNN classifier is developed afterwards to classify the tissue using either the DIC images or 2-D optical property maps of , and g. The DCNN classifier with the optical property maps exhibits high accuracy, significantly outperforming the same DCNN classifier on the DIC images. The label-free quantitative phase microscopy together with deep learning may emerge as a promising approach for in situ rapid cancer diagnosis.

Principal component analysis-based quantitative differential interference contrast microscopy.

Classical differential interference contrast (DIC) microscopy is an excellent tool for rendering high-contrast images to observe transparent specimen. Though the monochromatic shadow-cast image qualitatively reflects the specimen configuration, its phase can hardly be extracted, inevitably limiting its application in quantitative measurements. In order to extend its application, we propose a quantitative DIC (qDIC) microscopy technique, which can extract the specimen phase from phase-shifting beam-shearing interference images combining principal component analysis (PCA) and phase integration methods. Proved by both simulations and experiments, the PCA-based qDIC microscopy can rapidly and accurately retrieve the quantitative specimen phase. The new proposed technique shares with conventional DIC the advantage of high-contrast imaging, especially for transparent label-free specimens, but transforms DIC microscopy into a quantitative phase microscopy technique. We envision PCA-based qDIC microscopy as a future quantitative biological imaging modality.

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy.

We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100 × / 1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ? 0.5 ?? nm and lateral resolution if ? 300 ?? nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems.

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.

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

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.

Using liquid crystal variable retarders for fast modulation of bias and shear direction in quantitative differential interference contrast (DIC) microscope.

Conventional DIC microscope shows the two-dimensional distribution of optical path length gradient encountered along the shear direction between two interfering beams. It is therefore necessary to rotate unknown objects in order to examine them at several orientations. We built new DIC beam shearing assembly, which allows the bias to be modulated and shear directions to be switched rapidly without any mechanically rotating the specimen or the prisms. The assembly consists of two standard DIC prisms with liquid crystal cell in between. Another liquid crystal cell is employed for modulating a bias. All components do not require a special design and are available on the market. We describe techniques for measuring parameters of DIC prisms and calibrating liquid crystal cells. One beam-shearing assembly is added to the illumination path and another one to the imaging path of standard microscope. Two sets of raw DIC images at the orthogonal shear directions and two or three different biases are captured and processed within a second. Then the quantitative image of optical path gradient distribution within a thin optical section is displayed on a computer screen. The obtained data are also used to compute the quantitative distribution of optical phase, which represents refractive index gradient or height distribution. It is possible to generate back the enhanced regular DIC images with any desired shear direction. New DIC microscope can be combined with other techniques, such as fluorescence and polarization microscopy.

P‐58: Accelerating Phase Shifting Technique in Quantitative Differential Interference Contrast System for Critical Dimension Measurement of TFT Substrate

Abstract: A quantitative differential interference contrast (DIC) technique is proposed to measure the step height of TFT substrate, which is widely applied in display devices. Theoretical comparisons between different phase shifting techniques of DIC are given. Not only series of simulations are conducted but also the preliminary experiments of TFT dimension measurement are presented. The proposed accelerating phase shifting DIC technique might be adopted to topography measurement of optical components in industry.

Optical Quantification of Cellular Mass, Volume, and Density of Circulating Tumor Cells Identified in an Ovarian Cancer Patient

Clinical studies have demonstrated that circulating tumor cells (CTCs) are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal, and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD)-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC) compared to 18.7 ± 0.6 pg (leukocytes), p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC) compared to 230.9 ± 78.5 fL (leukocyte), p < 0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC) compared to 0.085 ± 0.004 pg/fL (leukocyte), p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.

Characterization of Single Platelet Mass, Volume, and Density in Response to Agonist Stimulation

Abstract 5261An increase in mean platelet volume (MPV) is correlated with platelet activation and subsequent shape change. Pathologic processes marked by increased platelet activity such as myocardial infarction, cerebral vascular accidents, diabetes mellitus, and hypertension are associated with an increased MPV. Elevated MPV in these conditions reflect both a higher level of platelet activation as well as increased platelet turnover secondary to platelet consumption within thrombus formation. Assessment of MPV can be used to risk stratify patients as well as assign them to prognostic categories. However, MPV does not assess platelet heterogeneity or the specific change in single platelet mass, volume, or density. Current methods provide little insight into changes in physical parameters at the single platelet level. In order to overcome this limitation, we developed a quantitative tomographic differential interference contrast (QTDIC) microscopy technique to measure dry mass, volume, and density of platelets at the single-cell level. This technique is based on determining the axially resolved refractive index from a series of through-focus DIC images. Single cell platelet mass was observed to reduce from 1.84 ± 0.14 pg to 1.60 ± 0.13 pg in response to stimulation with thrombin-receptor agonist peptide (TRAP), while single cell platelet volume reduced from 7.28 ± 0.56 fL to 6.03 ± 0.48 fL (mean ± SEM). Single cell platelet density increased from 0.25 ± 0.001 pg/fL to 0.26 ± 0.002 pg/fL (mean ± SEM). Taken together, we have characterized the physical parameters of platelets in response to agonist stimulation. Our data suggest that platelet activation may correlate with decreased mass and volume, perhaps as a consequence of platelet degranulation. Further elucidation of the morphological changes of activated platelets at the single platelet level may allow for better understanding of platelet function and dysfunction in patients affected by platelet granule deficiencies, giant platelet syndromes, and disorders associated with membrane receptors [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Quantitative analysis of shape and volume changes in activated thrombocytes in real time by single‐shot spatial light modulator‐based differential interference contrast imaging

We suggest to use a combination of optical tweezers and single-image quantitative differential interference contrast (DIC) emulated by a spatial light modulator (SLM) to study physiological shape changes in thrombocytes after activation and demonstrate the effectiveness of this system for the given task. A specially designed phase mask displayed at the SLM enables quantitative phase calculation from only a single recording. The optical tweezers stabilize trapped thrombocytes for long-time monitoring of changes in the optical thickness profile of thrombocytes during activation by adenosine diphosphate (ADP).

Quantitative DIC microscopy using an off-axis self-interference approach

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.