Phase Imaging Of Biological Cells Research Articles

Live Cell Imaging by Single-Shot Common-Path Wide Field-of-View Reflective Digital Holographic Microscope.

Quantitative phase imaging by digital holographic microscopy (DHM) is a nondestructive and label-free technique that has been playing an indispensable role in the fields of science, technology, and biomedical imaging. The technique is competent in imaging and analyzing label-free living cells and investigating reflective surfaces. Herein, we introduce a new configuration of a wide field-of-view single-shot common-path off-axis reflective DHM for the quantitative phase imaging of biological cells that leverages several advantages, including being less-vibration sensitive to external perturbations due to its common-path configuration, also being compact in size, simple in optical design, highly stable, and cost-effective. A detailed description of the proposed DHM system, including its optical design, working principle, and capability for phase imaging, is presented. The applications of the proposed system are demonstrated through quantitative phase imaging results obtained from the reflective surface (USAF resolution test target) as well as transparent samples (living plant cells). The proposed system could find its applications in the investigation of several biological specimens and the optical metrology of micro-surfaces.

Phase retrieval without phase unwrapping for white blood cells in deep-learning phase-shifting digital holography

Phase retrieval and phase unwrapping are the two important problems for enabling quantitative phase imaging of cells in phase-shifting digital holography. To simultaneously cope with these two problems, a deep-learning phase-shifting digital holography method is proposed in this paper. The proposed method can establish the continuous mapping function of the interferogram to the ground-truth phase using the end-to-end convolutional neural network. With a well-trained deep convolutional neural network, this method can retrieve the phase from one-frame blindly phase-shifted interferogram, without phase unwrapping. The feasibility and applicability of the proposed method are verified by the simulation experiments of the microsphere and white blood cells, respectively. This method will pave the way to the quantitative phase imaging of biological cells with complex substructures.

Quantitative phase imaging of biological cells using lensless inline holographic microscopy through sparsity-assisted iterative phase retrieval algorithm

The twin image-free phase reconstruction is still a challenge with single-shot inline holographic systems. Existing solutions mostly are based on the inverse problem approaches or alternating projections. However, there exists a trade-off between phase retrieval and twin image elimination. Recent studies have introduced a hybrid method involving both the approaches to mitigate this trade-off. Following these works, we propose a single-shot sparsity-assisted iterative phase retrieval approach that applies a sparsity constraint in the object domain and formulates phase retrieval as a minimization problem. We demonstrate lensless digital inline holographic microscopy for imaging transparent and weakly scattering biological samples over a large field-of-view of ∼29mm2. The proposed method achieves high fidelity phase reconstruction with faster convergence compared to the existing single-shot phase retrieval methods. We further demonstrate the phase quantification of label-free biological samples, such as cervical cells and RBCs, to highlight the potential of our technique in clinical applications.

Single-shot interference microscopy using a wedged glass plate for quantitative phase imaging of biological cells

To achieve quantitative phase imaging of biological cells, a new and simple single-shot interference microscopy is demonstrated by using a single rectangular wedged glass plate. This method is very compact, portable, and easy to implement. For this proposed interference microscopy, the wedged glass plate is used to deflect one half of the collimated incident beam toward another half. Subsequently, the two halves encounter and generate off-axis interference. Furthermore, based on the above system configuration, we also demonstrate dual channels interference successfully. For this situation, a common non-polarization cube beam-splitter is tilted and inset in front of the wedged glass plate. When the sample is only irradiated by one half of the incident beam while the incident beam is only incident to one side of the center semi-reflecting layer of the cube beam-splitter, two interference channels with a relative π (rad) phase-shift in one interferogram can be produced simultaneously. Using the proposed method, the phase information of the biological cell (unicellular paramecium) is obtained successfully. The experimental results show that our proposed method is practical and promising.

Quantitative and Dynamic Phase Imaging of Biological Cells by the Use of the Digital Holographic Microscopy Based on a Beam Displacer Unit

The digital holographic microscopy can be used for the quantitative and dynamic phase imaging of biological cells, which has a very wide application in the fields of biological and medical sciences. To make the measurement system simple, compact, and low-cost, a common-path configuration based on a beam displacer unit is introduced in the digital holographic microscopy. The simple optical structure and common-path design reduces the system requirement for the light source coherence, realizes the convenient adjustment of the object and reference beams and achieves an excellent system temporal stability of 0.53 nm. The living mouse osteoblastic cells during mitotic phase are quantitatively measured to demonstrate the capability and applicability of the system.

Dual-channel quantitative phase microscopy based on a single cube beamsplitter interferometer

Quantitative phase microscopy, as a non-destructive and non-invasive measurement technique, can indirectly reflect three-dimensional (3D) morphology and optical properties of transparent microstructure object by measuring phase information. In recent years, this kind of technique has been widely used to detect and investigate the characteristics of biological cells and it has become more and more important in the field of modern biomedical and life science. In this paper, only by using a single cube beamsplitter interferometer, a simple single-shot dual-channel quantitative phase microscopic measurement technique is demonstrated for 3D quantitative phase imaging of biological cells. In the proposed method, a conventional non-polarized cube beamsplitter is the most pivotal element. Unlike its traditional application method, the cube beamsplitter is tilted in a nonconventional configuration and the illumination beam is only incident on the left (or right) half of the cube beamsplitter (just the one side of central semi-reflecting layer), and a very small angle is introduced between the central semi-reflecting layer and the optical axis of incident beam. Based on the light splitting characteristic of the cube beamsplitter, two replicas of incident beam are generated. These two generated replicas (transmission beam and reflection beam) are of symmetry with respect to each other, and they will encounter and form interference when the direction of the incident beam meets a certain condition. Adjust the sample to a suitable position and make it only contact one half of incident beam, and the modulated beam will be seen as the object beam and the remaining clean half of incident beam as the reference beam. When the interference phenomenon occurs, two interference channels with a relative π (rad) phase-shift in one interferogram are acquired simultaneously only using one digital camera, and the higher spatial frequency of interference fringes can be achieved by adjusting a relatively big angle between the central semi-reflecting layer and the optical axis of incident beam. Because of the off-axis interference mode, we only need to record one interferogram to gain the continuous phase information and avoid using complex phase-shift techniques. At the same time, this proposed method is of simple structure and easy to operate due to using less ordinary off-the-shelf optical elements. All these simplify the structure of the system and reduce the cost of the system as much as possible. Finally, the phase information of paramecium is successfully obtained from different interference channels respectively. Furthermore, according to the characteristic of π (rad) phase-shift, we also realize the calibration and determination of ultimate precise phase information of sample by using the method of averaging between these two channels. The experimental results show that our proposed method is suitable for 3D surface morphology measurement of small transparent samples.

Optimal spatial bandwidth capacity in multiplexed off-axis holography for rapid quantitative phase reconstruction and visualization

We present new methods for optimizing the spatial bandwidth capacity in off-axis holography using spatial multiplexing. We use optimal spatial multiplexing of off-axis holograms to fill the entire spatial frequency domain, including the space previously occupied by the intensity of the sample. Our approach enables spatial digital compression of eight off-axis holograms into a single real-valued multiplexed hologram, having the same number of pixels as each of the input holograms, but still allowing their full reconstruction without resolution or magnification loss in the reconstructed complex wave fronts. This new method allows 33% improvement in usage of the spatial bandwidth capacity compared to the best available off-axis holography real-value multiplexing method. Since the output multiplexed hologram contains only real values, it can be used for rapid display of eight wave front reconstructions at once, which is useful for real-time visualization, when the hologram display device is slower than the acquiring camera. We further generalize this technique to digital multiplexing of 16 real-valued holograms into a single complex-valued hologram by simple arithmetic operations in the hologram domain. Then, the extraction of the 16 wave fronts includes a single 2-D discrete Fourier transform to access the spatial frequency domain, allowing fast reconstruction, which is useful for real-time processing of off-axis holograms, with improved processing rate compared to current hologram processing algorithms. These new approaches allow the full reconstruction of all compressed data without loss of resolution or magnification, even though the samples are dense such that their frequency content employs the entire range. Both multiplexing architectures are then demonstrated for experimentally-acquired off-axis holograms for quantitative phase imaging of biological cells.

Dual-wavelength common-path digital holographic microscopy for quantitative phase imaging of biological cells

Biological cells are usually transparent with a small refractive index gradient. Digital holographic interferometry can be used in the measurement of biological cells. We propose a dual-wavelength common-path digital holographic microscopy for the quantitative phase imaging of biological cells. In the proposed configuration, a parallel glass plate is inserted in the light path to create the lateral shearing, and two lasers with different wavelengths are used as the light source to form the dual-wavelength composite digital hologram. The information of biological cells for different wavelengths is separated and extracted in the Fourier domain of the hologram, and then combined to a shorter wavelength in the measurement process. This method could improve the system’s temporal stability and reduce speckle noises simultaneously. Mouse osteoblastic cells and peony pollens are measured to show the feasibility of this method.

Angular phase unwrapping of optically thick objects with a thin dimension.

We present a new phase unwrapping approach, which allows reconstruction of optically thick objects that are optically thin from at least one viewing angle, by considering the information stored in the object phase maps captured from consecutive angles. Our algorithm combines 1-D phase unwrapping in the angular dimension with conventional 2-D phase unwrapping, to achieve unwrapping of the object from the optically thick perspective. We thus obtain quantitative phase imaging of objects that were previously impossible to image in certain viewing angles. To demonstrate our approach, we present both numerical simulation and experimental results for quantitative phase imaging of biological cells.

Quantitative phase imaging of biological cells using spatially low and temporally high coherent light source.

In this Letter, we demonstrate quantitative phase imaging of biological samples, such as human red blood cells (RBCs) and onion cells using narrow temporal frequency and wide angular frequency spectrum light source. This type of light source was synthesized by the combined effect of spatial, angular, and temporal diversity of speckle reduction technique. The importance of using low spatial and high temporal coherence light source over the broad band and narrow band light source is that it does not require any dispersion compensation mechanism for biological samples. Further, it avoids the formation of speckle or spurious fringes which arises while using narrow band light source.