Digital holographic (DH) microscopy has grown into a powerful nondestructive technique for real-time study of living cells including dynamic membrane changes and cell fluctuations in nanometer and sub-nanometer scales. The conventional DH microscopy configurations require a separately generated coherent reference wave that results in a low phase stability and a necessity to precisely adjust the intensity ratio between two overlapping beams. In this work, we present a compact, simple and very stable common-path DH microscope, employing a self-referencing configuration. The microscope is implemented by a diode laser as the source and a Fresnel biprism for splitting and recombining the beams simultaneously. In the overlapping area, linear interference fringes with high contrast are produced. The frequency of the interference pattern could be easily adjusted by displacement of the biprism along the optical axis without decrease in fringe contrast. To evaluate the validity of the method, the spatial noise and temporal stability of the setup are compared with the common off-axis DH microscope based on Mach-Zehnder interferometer (MZI). It is shown that the proposed technique has low mechanical noise as well as superb temporal stability with sub-nanometer precision without any external vibration isolation. The higher temporal stability improves the capabilities of the microscope for studying micro-objects fluctuations, particularly in the case of biological specimens. Experimental results are presented using red blood cells (RBCs) and silica microspheres to demonstrate the system performance.

We present an integrated optical system for three-dimensional (3D) imaging of micrometer-sized samples, while immobilizing and manipulating the samples by means of an optical fiber trap. Optical traps allow us to apply and measure pico-Newton-sized forces, and perform detailed measurements of micrometer-sized dielectric systems in the field of biology. The integrated 3D system can be used as a major tool in the field of biophysics. The trap is built using a tapered optical fiber to enhance the effective numerical aperture of the fiber. The trapping system is mounted on a conventional microscope, in which the two eyepieces' output ports are used as the paths of an off-axis self-referencing digital holographic microscopy (DHM) setup. The trap is calibrated using a high-speed camera, and trap stiffness is determined through the power spectrum method. The compact setup provides an elegant apparatus for temporally stable DHM for 3D imaging of optically controlled samples. Three-dimensional information and quantitative phase contrast images of the trapped samples are obtained by postprocessing the recorded digital holograms. Experiments were performed on lipids and red blood cells. Quantitative phase contrast images and temporal evolution of optical thickness of trapped samples are presented.

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Phase-shifting digital holography is widely considered to be a groundbreaking method to quantitatively investigate the phase distribution of specimens, such as living cells. The main flaws of this method, however, are that the requirement for several sequential phase-shifted holograms eliminates the possibility of single-shot imaging and complex configurations would also increase the temporal noise. The present paper aims to validate a single-shot, common-path, phase-shifting digital holographic microscopy, employing a self-referencing geometry. A Ronchi ruling, located in the Fourier plane of a standard microscopic imaging system, produces multiple replicas of sample information in the image plane. The phase retrieval algorithm is performed by superposition of the sample-free portion of each replica with the object information, and requires at least three adjacent diffraction orders. To evaluate the performance of the proposed method, the phase distribution of silica microspheres as a test sample and the morphology of red blood cells as a biological specimen are determined. This configuration offers improved temporal stability in comparison with previously reported Mach–Zehnder interferometers, and may serve as an alternative for real-time surveying of nanometric and subnanometric fluctuations of living microscopic specimens.

The structural complexity and instability of many interference phase microscopy methods are the major obstacles toward high-precision phase measurement. In this vein, improving more efficient configurations as well as proposing new methods are the subjects of growing interest. Here we introduce Fresnel diffraction from a phase step to the realm of quantitative phase imaging. By employing Fresnel diffraction of a divergent (or convergent) beam of light from a plane-parallel phase plate, we provide a viable, simple and compact platform for three-dimensional imaging of micron-sized specimens. The recorded diffraction pattern of the outgoing light from an imaging system in the vicinity of the plate edge can be served as a hologram, which would be analyzed via Fourier transform method to measure the sample phase information. The period of diffraction fringes is adjustable simply by rotating the plate without the reduction of both field of view and fringe contrast. The high stability of the presented method is affirmatively confirmed through comparison the result with that of conventional Mach-Zehnder based digital holographic method. Quantitative phase measurements on silica microspheres, onion skin and red blood cells ensure the validity of the method and its ability for monitoring nanometer-scale fluctuations of living cells, particularly in real-time.

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In this Letter, a very simple, stable, and portable lensless digital holographic (DH) microscopy method is presented relying on the Fresnel diffraction (FD) of light from a phase discontinuity (PD). A phase plate in the transmission or a physical step in the reflection can be employed in the path of the divergent beam of a coherent light source as a component imposing the PD. The recorded diffraction pattern in the vicinity of the PD is a hologram produced by off-axis overlapping of two diffracted waves in both sides of the boundary region with adjustable fringe modulation. To validate the method, measurements are performed on the amplitude and phase specimens as well as on the dynamic processes of water evaporation and 3D tracking of floating cells. A reflective configuration of FD from a physical step can be used as a powerful platform for lensless DH microscopy using high-energy electromagnetic radiation, e.g., x-ray and UV sources for the high-resolution imaging of moving samples.

One of the most dangerous human pathogens with high prevalence worldwide is Streptococcus pyogenes, which has major impacts on global morbidity and mortality. A major challenge for S. pyogenes vaccine development is the detection of epitopes that confer protection from infection by multiple S. pyogenes types. Our aim was to identify the most conserved and immunogenic antigens of S. pyogenes, which can be a potential candidate for vaccine design in the future. Eight important surface proteins were analyzed. Using different prediction servers, strongest epitopes were selected. They had the ability to stimulate the humoral and cell-mediated immune system. Molecular docking was performed for measuring free-binding energy of selected epitopes. Seven epitopes from three surface proteins were selected as potential candidates for vaccine development. Conservation of selected epitopes among different Streptococcus types was checked. Further in vitro and in vivo tests are required to validate the suitability of the epitopes for vaccine design.