Stable and simple quantitative phase-contrast imaging by Fresnel biprism (original) (raw)
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Optics Letters, 2012
Digital holographic microscopy (DHM) is one of the most effective techniques used for quantitative phase imaging of cells. Here we present a compact, easy to implement, portable, and very stable DHM setup employing a selfreferencing Lloyd's mirror configuration. The microscope is constructed using a diode laser source and a CMOS sensor, making it cost effective. The reconstruction of recorded holograms yields the amplitude and phase information of the object. The temporal stability of the presented technique was found to be around 0.9 nm without any vibration compensation, which makes it ideal for studying cell profile changes. This aspect of the technique is demonstrated by studying membrane fluctuations of red blood cells.
Structured illumination in Fresnel biprism-based digital holographic microscopy
Optics and Lasers in Engineering
The structured illumination (SI) architecture can be adopted in a digital holographic (DH) microscope to enhance the spatial resolution. In this paper, we propose and demonstrate a compact and simple SI method in a self-referencing common-path configuration of DH microscopy. The combination of SI pattern generated by a compact disk and common-path off-axis geometry formed by Fresnel biprism introduces a low-cost and highly stable system to image both amplitude and phase objects with a twofold improvement of the spatial resolution. Experimental results for the case of a standard test sample validate the predicted resolution enhancement compared to that of the conventional imager. The presented DH method allows for imaging living cells with strikingly improved clarity compared to the conventional DH microscopes.
Quantitative Phase Imaging IV, 2018
Main restrictions of using laser light in digital holographic microscopy (DHM) are coherence induced noise and parasitic reflections in the experimental setup which limit resolution and measurement accuracy. We explored, if coherence properties of partial coherent light sources can be generated synthetically utilizing spectrally tunable lasers. The concept of the method is demonstrated by label-free quantitative phase imaging of living pancreatic tumor cells and utilizing an experimental configuration including a commercial microscope and a laser source with a broad tunable spectral range of more than 200 nm.
Common-path lensless digital holographic microscope employing a Fresnel biprism
Optics and Lasers in Engineering, 2020
Digital holographic (DH) microscopy is a nondestructive quantitative method for studying micron-sized samples with a special application in medicine, biology and materials science. Off-axis geometry of this method generates high-quality quantitative phase images employing beam-splitters, mirrors, condensers and magnifying lenses, which greatly increase the cost, size and sensitivity of the system against mechanical noise effects. The essential aim of recently proposed common-path configurations is to make viable approaches which minimize the mechanical and optical noises. Here we present a lensless DH microscopy system based on a common-path geometry using a Fresnel biprism for formation of off-axis geometry. The system comprises a lens for producing two spherical waves and by carefully choosing its parameters, we can construct a 3D imaging microscope with desired fringe spacing and overlapping region which is employed for studying many samples with different sizes, shapes and confluences. The system is compact, lightweight and robust versus mechanical vibrations. The ability to image amplitude and phase samples has been tested using standard test samples, living cells and water droplets.
High-resolution quantitative phase-contrast microscopy by digital holography
Optics Express, 2005
Techniques of digital holography are improved in order to obtain highresolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating holographic optical field is seen to have significant advantages including tight control of spurious noise components. Holographic phase images are obtained with 0.5 μm diffraction-limited lateral resolution and largely immune from the coherent noise common in other holographic techniques. The phase profile is accurate to about 30 nm of optical thickness. Images of SKOV-3 ovarian cancer cells display intracellular and intranuclear organelles with clarity and quantitative accuracy.
Phase imaging of cells by simultaneous dual-wavelength reflection digital holography
Optics Express, 2008
We present a phase-imaging technique to quantitatively study the three-dimensional structure of cells. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of SKOV-3 ovarian cancer cells are presented.
Optics Letters, 2005
We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.
Applied Optics, 1999
We present a digital method for holographic microscopy involving a CCD camera as a recording device. Off-axis holograms recorded with a magnified image of microscopic objects are numerically reconstructed in amplitude and phase by calculation of scalar diffraction in the Fresnel approximation. For phasecontrast imaging the reconstruction method involves the computation of a digital replica of the reference wave. A digital method for the correction of the phase aberrations is presented. We present a detailed description of the reconstruction procedure and show that the transverse resolution is equal to the diffraction limit of the imaging system.
Common-path, single-shot phase-shifting digital holographic microscopy using a Ronchi ruling
Applied Physics Letters, 2019
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 a...
Applied Optics, 2003
An approach is proposed for removing the wave front curvature introduced by the microscope imaging objective in digital holography, which otherwise hinders the phase contrast imaging at reconstruction planes. The unwanted curvature is compensated by evaluating a correcting wave front at the hologram plane with no need for knowledge of the optical parameters, focal length of the imaging lens, or distances in the setup. Most importantly it is shown that a correction effect can be obtained at all reconstruction planes. Three different methods have been applied to evaluate the correction wave front and the methods are discussed in detail. The proposed approach is demonstrated by applying digital holography as a method of coherent microscopy for imaging amplitude and phase contrast of microstructures.