Holographic microscopy in low coherence

Differential phase contrast (DPC) is a non-interferometric quantitative phase imaging method achieved by using an asymmetric imaging procedure. We report a pupil modulation differential phase contrast (PMDPC) imaging method by filtering a sample's Fourier domain with half-circle pupils. A phase gradient image is captured with each half-circle pupil, and a quantitative high resolution phase image is obtained after a deconvolution process with a minimum of two phase gradient images. Here, we introduce PMDPC quantitative phase image reconstruction algorithm and realize it experimentally in a 4f system with an SLM placed at the pupil plane. In our current experimental setup with the numerical aperture of 0.36, we obtain a quantitative phase image with a resolution of 1.73μm after computationally removing system aberrations and refocusing. We also extend the depth of field digitally by 20 times to ±50μm with a resolution of 1.76μm.

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm2, corresponding to a space–bandwidth–time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

Quantitative phase imaging (QPI) became an important technique for label-free biomedical imaging suitable particularly for observation of live cell and dry-mass profiling. Extension of this technique to objects immersed in turbid medium is highly desirable with respect to the need of non-invasive observation of live cells in real 3D environments. Coherencecontrolled holographic microscopy is capable of QPI through turbid media owing to coherence gating induced in transmitted-light geometry by low spatial coherence of illumination. Using this approach, QPI of object in turbid medium can be formed both by ballistic and multiply scattered photons. Moreover, the particular QPIs formed by ballistic and scattered photons can be superimposed thus yielding synthetic QPI of substantially improved image quality. We support the theoretical reasoning of the effect by experimental data.

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Fluorescence microscopy is a golden standard for contemporary biological studies. However, since fluorescent dyes cross-react with biological processes, a label-free approach is more desirable. The aim of this study is to create artificial, fluorescence-like nuclei labeling from label-free images using Convolution Neural Network (CNN), where training data are easy to obtain if simultaneous label-free and fluorescence acquisition is available. This approach was tested on holographic microscopic image set of prostate non-tumor tissue (PNT1A) and metastatic tumor tissue (DU145) cells. SegNet and U-Net were tested and provide “synthetic” fluorescence staining, which are qualitatively sufficient for further analysis. Improvement was achieved with addition of bright-field image (by-product of holographic quantitative phase imaging) into analysis and two step learning approach, without and with augmentation, were introduced. Reconstructed staining was used for nucleus segmentation where 0.784 and 0.781 dice coefficient (for DU145 and PNT1A) were achieved.

As a typical computational method, Fourier ptychographic microscopy (FPM) can realize high spatial resolution and quantitative phase imaging while preserving the large field of view with a low numerical aperture (NA) objective. A programmable light-emitting diode (LED) array is used as a typical illuminator in an FPM system, and the illumination parameters of each LED element are crucial to the success of the FPM reconstruction algorithm. Compared with LED arrays arranged in rectangular arrays, LED arrays with special structures such as domes or rings can effectively improve FPM imaging results and imaging efficiency. As a trade-off, their calibration difficulty is greatly increased due to the lack of geometric constraints of rectangular arrays. In this paper, we propose an effective hybrid full-pose parameter calibration method for freeform LED array illuminators, combining stereoscopic 3D imaging techniques and the geometric constraints of the microscopic platform. First, a stereovision system is used to obtain the accurate 3D position of each LED element of the freeform illuminator and to construct a rigid 3D coordinate LED array system. Then, calibration between the coordinate system of the LED array and that of the optical imaging component is realized according to the geometric features of the brightfield-to-darkfield edges. Finally, we verify the feasibility and effectiveness of the proposed method through full-pose parameter calibration of LED arrays with different arrangement rules.

Imaging in turbid media is a challenging problem in biomedical and diagnosis fields. Coherence-Controlled Holographic Microscope design is capable of quantitative phase imaging in turbid media by coherence gating induced by incoherent illumination.

Investigating the crystalline structure and structural heterogeneity of starch granules using polarization-based quantitative phase microscopy.

In digital holographic microscopy (DHM) scattering patterns and parasitic interference fringes due to internal reflections that are caused by coherent laser light affect quantitative phase imaging (QPI). We present an approach for the reduction coherence induced disturbances. Therefore, the sample illumination is modulated by an electrically focus tunable lens while a series of digital off-axis holograms is recorded from which subsequently averaged QPI images are retrieved. The principle is compatible with Mach-Zehnder interferometer-based off-axis DHM and capable to be integrated into commercial research microscopes. The performance of our approach is demonstrated by results from living porcine epithelia cells and mouse fibroblasts.

Embolization of thrombotic material may lead to acute events such as ischemia and myocardial infarction. The embolus is the physical detachment from a primary thrombus that has developed under fluid shear rates. The physical characteristics (surface area coverage, volume, mass, and packing density) of a thrombus influence the overall flow dynamics of an occluding blood vessel. Here, the effectiveness of holographic quantitative phase microscopy (QPM) in identifying multiple morphological parameters of a thrombus (volume, surface area, and height) formed over collagen‐coated microfluidic channels by exerting a range of shear rates with anticoagulated platelet‐rich plasma (PRP) and whole blood is demonstrated. QPM enables the recording of entire thrombus volumes in real‐time using PRP and observed both growth and contraction trends of thrombi, without need for biochemical labeling. The process of emboli detachment in a microfluidic channel under pathophysiological shear rates (7500 and 12 500 s−1) is quantified. Rapid and direct quantification of an embolizing thrombus can enable the study of events during undesirable vessel occlusion and lead to targeting and early diagnosis of acute coronary and venous events.

Quantitative phase spectroscopy is presented as a novel method of measuring the wavelength-dependent refractive index of microscopic volumes. Light from a broadband source is filtered to an ~5 nm bandwidth and rapidly tuned across the visible spectrum in 1 nm increments by an acousto-optic tunable filter (AOTF). Quantitative phase images of semitransparent samples are recovered at each wavelength using off-axis interferometry and are processed to recover relative and absolute dispersion measurements. We demonstrate the utility of this approach by (i) spectrally averaging phase images to reduce coherent noise, (ii) measuring absorptive and dispersive features in microspheres, and (iii) quantifying bulk hemoglobin concentrations by absolute refractive index measurements. Considerations of using low coherence illumination and the extension of spectral techniques in quantitative phase measurements are discussed.

We present a method for observing complex amplitude fields by using a single-pixel camera without reference light and imaging optics. In the method, an object is illuminated with coherent light, and the propagating field is captured in multiple intensity measurements using a single photodetector and a spatial light modulator upon which different patterns are sequentially displayed. The object field is reconstructed from the intensity measurements with a compressive-sensing-based phase retrieval algorithm. Quantitative amplitude and phase imaging based on our method is experimentally demonstrated. In the experiments, objects were reconstructed from less than 10% of the number of measurements determined with conventional approaches.

Fourier Ptychography is a novel imaging technique consists in combining many coherent images acquired with various illumination angle and combined using an efficient phase retrieval algorithm. This technique allows to synthesize a larger numerical aperture than physically possible by the lens aperture alone, often limited by manufacturing capabilities. We present here an adaptation of the Fourier Ptychography technique to a synchrotron-based full-field microscope (SHARP) operating at 13.5nm (EUV wavelength) and demonstrate 26nm coherent resolution on reflective samples, alongside with quantitative phase imaging which allows to characterize sub-nanometer substrate roughness.

The advantages of quantitative phase microscopy (QPM) such as label-free imaging with high spatial sensitivity, live cell compatibility and high-speed imaging makes it viable for various biological applications. The measurement accuracy of QPM strongly relies on the shape of the recorded interferograms, whether straight or curved fringes are recorded during the data acquisition. Moreover, for a single shot phase recovery high fringe density is required. The wavefront curvature for the high-density fringes over the entire field of view is difficult to be discerned with the naked eye. As a consequence, there is a quadratic phase aberration in the recovered phase images due to curvature mismatch. In the present work, we have implemented sampling moiré method for real-time sensing of the wavefront curvature mismatch between the object and the reference wavefronts and further for its correction. By zooming out the interferogram, moiré fringes are generated which helps to easily identify the curvature of the fringes. The wavefront curvature mismatch correction accuracy of the method is tested with the help of low temporal coherent light source such as a white light (temporal coherence ∼ 1.6 µm). The proposed scheme is successfully demonstrated to remove the quadratic phase aberration caused due to wavefront mismatch from an USAF resolution target and the biological tissue samples. The phase recovery accuracy of the current scheme is further compared with and found to better than the standard method called principle component analysis. The proposed method enables recording of the corrected wavefront interferogram without needing any additional optical components or modification and also does not need any post-processing correction algorithms. The proposed method of curvature compensation paves the path for a high-throughput and accurate quantitative phase imaging.

Quantitative phase imaging (QPI) technique is used to determine various biophysical parameters, such as refractive index, cell thickness, morphology, etc. On the other hand, fluorescence microscopy is used to acquire information regarding molecular specificity of the biological cells and tissues. Conventionally, a fully coherent light source such as laser is used in QPI technique to obtain the interference fringes with ease; however, its high coherence is also responsible for the generation of speckle and spurious fringes, which results in degraded image quality and affects the phase measurement results too. In this paper, we report a multi-modal system that can be effectively utilized to acquire time varied diverse information about the biological specimen with high spatial phase sensitivity. Herein, a single unit comprising of a fluorescence microscope and the Linnik based interferometer specially equipped with a partially spatially coherent light source illumination was developed. The integrated system is capable to procure molecular specificity and phase information of biological specimen, in a single shot, utilizing a single-chip color CCD camera. Here, we performed experiments on MG63 osteosarcoma cells, and the composite interferometric-fluorescence images were obtained and then digitally decomposed into red and green colors; and, the phase maps were reconstructed using the Fourier fringe analysis method. Furthermore, the cultured cells were monitored over a time-span to observe and investigate the time dependent morphological changes along with the quantification of cellular adhesion and spreading. Hence, the proposed system can be utilized to quantify time dependent changes in the cell’s morphology and in cell adhesion which can be an indicator for the detection of various range of diseases such as arthritis, cancer, osteoporosis and atherosclerosis.