Digital in-line holographic microscopy

We first briefly review the state of the art of digital in-line holographic microscopy (DIHM) with numerical reconstruction and then discuss some technical issues, such as lateral and depth resolution, depth of field, twin image, four-dimensional tracking, and reconstruction algorithm. We then present a host of examples from microfluidics and biology of tracking the motion of spheres, algae, and bacteria. Finally, we introduce an underwater version of DIHM that is suitable for in situ studies in an ocean environment that show the motion of various plankton species.     

Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging

Digital holography enables a multifocus quantitative phase microscopy for the investigation of reflective surfaces and for marker-free live cell imaging. For digital holographic long-term investigations of living cells an automated (subsequent) robust and reliable numerical focus adjustment is of particular importance. Four numerical methods for the determination of the optimal focus position in the numerical reconstruction and propagation of the complex object waves of pure phase objects are characterized, compared, and adapted to the requirements of digital holographic microscopy. Results from investigations of an engineered surface and human pancreas tumor cells demonstrate the applicability of Fourier-weighting- and gradient-operator-based methods for robust and reliable automated subsequent numerical digital holographic focusing.     

Digital holographic microscopy with dual-wavelength phase unwrapping

We apply the techniques of digital holography to obtain microscopic three-dimensional images of biological cells. The optical system is capable of microscopic holography with diffraction-limited resolution by projecting a magnified image of a microscopic hologram plane onto a CCD plane. Two-wavelength phase-imaging digital holography is applied to produce unwrapped phase images of biological cells. The method of three-wavelength phase imaging is proposed to extend the axial range and reduce the effect of phase noise. These results demonstrate the effectiveness of digital holography in high-resolution biological microscopy.     

Digital holographic microscopy with pure-optical spherical phase compensation

Telecentric architecture is proposed for circumventing, by the pure-optical method, the residual parabolic phase distortion inherent to standard configuration of digital holographic microscopy. This optical circumvention produces several important advantages. One is that there is no need for computer compensation of the parabolic phase during the phase map recovering procedure. The other is that in off-axis configuration, the spatial frequency useful domain is enlarged. The validity of the method is demonstrated by performing quantitative measurement of depth differences with high axial resolution.     

Digital holographic microscopy with reduced spatial coherence for three-dimensional particle flow analysis

We investigate the use of a digital holographic microscope working in partially coherent illumination to study in three dimensions a micrometer-size particle flow. The phenomenon under investigation rapidly varies in such a way that it is necessary to record, for every camera frame, the complete holographic information for further processing. For this purpose, we implement the Fourier-transform method for optical amplitude extraction. The suspension of particles is flowing in a split-flow lateral-transport thin separation cell that is usually used to separate the species by their sizes. Details of the optical implementation are provided. Examples of reconstructed images of different particle sizes are shown, and a particle-velocity measurement technique that is based on the blurred holographic image is exploited.     

Automatic procedure for aberration compensation in digital holographic microscopy and applications to specimen shape compensation

We present a procedure that compensates for phase aberrations in digital holographic microscopy by computing a polynomial phase mask directly from the hologram. The phase-mask parameters are computed automatically without knowledge of physical values such as wave vectors, focal lengths, or distances. This method enables one to reconstruct correct and accurate phase distributions, even in the presence of strong and high-order aberrations. Examples of applications are shown for microlens imaging and for compensating for the deformations associated with a tilted thick plate. Finally we show that this method allows compensation for the curvature of the specimen, revealing its surface defects and roughness. Examples of applications are shown for microlenses and metallic sphere imaging.     

Digital holographic microscopy in the deep (193 nm) ultraviolet

We present a system based on digital holography suitable for the investigation of microscopic objects. To increase the resolution of the system a deep (193 nm) UV laser source has been used. A method for compensating aberrations due to the non-perfect optical elements used for the recording has been developed. The system allows the investigation of reflecting and transmitting samples.     

Digital holographic microscopy with sequential off-axis illumination directions based on spatial light modulator

Digital holographic microscopy (DHM) with time-sequential off-axis illumination directions can synthesize a large aperture, and thus have a higher spatial resolution than the one with on-axis illumination. In this paper, time-sequential off-axis illumination directions are generated by a spatial light modulator (SLM) in DHM, and the residual phases along different illumination directions are suppressed by using the phase compensation technique, as such the image with resolution enhancement is reconstructed. The usage of SLM enables shifting the illumination for different orientations and phase shifts without mechanical motion. The experiments have been conducted to verify the feasibility of this method on the residual phase suppression.     

Space-bandwidth conditions for efficient phase-shifting digital holographic microscopy

Microscopy by holographic means is attractive because it permits true three-dimensional (3D) visualization and 3D display of the objects. We investigate the necessary condition on the object size and spatial bandwidth for complete 3D microscopic imaging with phase-shifting digital holography with various common arrangements. The cases for which a Fresnel holographic arrangement is sufficient and those for which object magnification is necessary are defined. Limitations set by digital sensors are analyzed in the Wigner domain. The trade-offs between the various holographic arrangements in terms of conditions on the object size and bandwidth, recording conditions required for complete representation, and complexity are discussed.     

Biological scanning electrochemical microscopy and its application to live cell studies

Progress in instrumental design continues to pave the way for high-resolution, real-time electrochemical measurements with living cells.     

High-speed holographic microscopy for fast-propagating cracks in transparent materials

A method of high-speed holographic microscopy is developed to take three successive microscopic photographs of a crack tip propagating in a transparent poly(methyl methacrylate) specimen at a speed of several hundred meters per second. When a crack is propagating in a specimen, three Q-switched ruby lasers emit three laser pulses successively. The time interval between each laser pulse and the next is 1 mus or longer. An optical system of angle-multiplexing holography records the crack as three successive holograms on one photographic plate. Crack images are reconstructed and photographed through a conventional microscope. The spatial resolution of the reconstructed images is approximately 114 lines/mm. From the photographs, one can measure crack speed, crack opening displacement, and the dynamic stress intensity factor. The high-speed holographic microscopy makes it possible to study rapid crack propagation in microseconds.     

Fast numerical reconstruction technique for high-resolution hybrid holographic microscopy

A numerical reconstruction method believed to be new is proposed for hybrid holographic microscopy in which the hologram of a microscopic object is recorded by an image sensor and is then reconstructed by a computer. Because the Fresnel-Kirchhoff integral must be used for numerical reconstruction to achieve high resolution, we propose an approximation technique for reducing the calculation time. This approximation technique is suitable for microscopic application. The numerical reconstruction of 1-mum-sized objects was demonstrated with a He-Ne laser (lambda = 0.6328 mum).     

Endoscopic double-pulse electronic-speckle-pattern interferometer for technical and medical intracavity inspection

An endoscope electronic-speckle-pattern interferometer (ESPI) camera system is presented that can be applied to examinations of technical objects as well as for in vitro and in vivo minimal invasive medical diagnostics. Integration of optical fibers for the guidance of a cw-laser beam and an endoscopic imaging system yield a compact ESPI system that opens up new possibilities for highly sensitive interferometric intracavity inspection under handheld conditions. A CCD camera in combination with a fast frame-grabber system allows dynamic image subtractions at a frequency rate of as much as 25 Hz with high fringe contrast. Results from investigations of technical objects and biological objects in vitro and in vivo are obtained. In endoscopic minimal invasive therapy this method could substitute for the missing operator's tactile contact with the treated tissue by replacing it with visual information (endoscopic taction).     

Digital holographic microscope for measuring three-dimensional particle distributions and motions

Better understanding of particle-particle and particle-fluid interactions requires accurate 3D measurements of particle distributions and motions. We introduce the application of in-line digital holographic microscopy as a viable tool for measuring distributions of dense micrometer (3.2 microm) and submicrometer (0.75 microm) particles in a liquid solution with large depths of 1-10 mm. By recording a magnified hologram, we obtain a depth of field of approximately 1000 times the object diameter and a reduced depth of focus of approximately 10 particle diameters, both representing substantial improvements compared to a conventional microscope and in-line holography. Quantitative information on depth of field, depth of focus, and axial resolution is provided. We demonstrate that digital holographic microscopy can resolve the locations of several thousand particles and can measure their motions and trajectories using cinematographic holography. A sample trajectory and detailed morphological information of a free-swimming copepod nauplius are presented.     

Fluid surface compensation in digital holographic microscopy for topography measurement

A novel technique is presented for surface compensation and topography measurement of a specimen in fluid medium by digital holographic microscopy (DHM). In the measurement, the specimen is preserved in a culture dish full of liquid culture medium and an environmental vibration induces a series of ripples to create a non-uniform background on the reconstructed phase image. A background surface compensation algorithm is proposed to account for this problem. First, we distinguish the cell image from the non-uniform background and a morphological image operation is used to reduce the noise effect on the background surface areas. Then, an adaptive sampling from the background surface is employed, taking dense samples from the high-variation area while leaving the smooth region mostly untouched. A surface fitting algorithm based on the optimal bi-cubic functional approximation is used to establish a whole background surface for the phase image. Once the background surface is found, the background compensated phase can be obtained by subtracting the estimated background from the original phase image. From the experimental results, the proposed algorithm performs effectively in removing the non-uniform background of the phase image and has the ability to obtain the specimen topography inside fluid medium under environmental vibrations.     

Partially coherent light-emitting diode illumination for video-rate in-line holographic microscopy

The light of a light-emitting diode or a common thermal source, such as a tungsten filament lamp, is known to be quasi-incoherent. We generated partially coherent light of these sources with a volume of coherence in the micrometer range of 5-100 μm3 by spatial and spectral filtering. The corresponding degree of partial coherence was adapted for microscopic interference setups, such as a digital in-line holographic microscope. The practicability of the sources was determined by the spectral emittance and the resulting signal-to-noise ratio (SNR) of the detector. The microscale coherence in correlation with the SNR and its resolution for microscopy were analyzed. We demonstrate how low-light-level, non-laser sources enable holographic imaging with a video frame rate (25 frames/s), an intermediate SNR of 8 dB, and a volume of coherence of 3.4×10(4) μm3. Holograms of objects with a lateral resolution of 1 μm were achieved using a microscope lens (50×/NA=0.7) and a CCD camera featuring a 4-12 bit dynamic range.     

Hybrid holographic microscopy free of conjugate and zero-order images

The true image area that can be used for recording microscopic objects in hybrid holographic microscopy can be increased by elimination of the conjugate image and the zero-order image. We therefore added two shutters and one phase modulator to the electro-optical holographic recording system so that we could change the recording parameters and evaluate four methods of eliminating the conjugate image and the zero-order image. We found that the methods that use only the phase modulator require the recording of fewer holograms than do the methods that use the shutters and also provide reconstructed images that are less noisy.     

Threshold automatic selection hybrid phase unwrapping algorithm for digital holographic microscopy

Conventional quality-guided (QG) phase unwrapping algorithm is hard to be applied to digital holographic microscopy because of the long execution time. In this paper, we present a threshold automatic selection hybrid phase unwrapping algorithm that combines the existing QG algorithm and the flood-filled (FF) algorithm to solve this problem. The original wrapped phase map is divided into high- and low-quality sub-maps by selecting a threshold automatically, and then the FF and QG unwrapping algorithms are used in each level to unwrap the phase, respectively. The feasibility of the proposed method is proved by experimental results, and the execution speed is shown to be much faster than that of the original QG unwrapping algorithm.