Submicrometer optical tomography by multiple-wavelength digital holographic microscopy

We present a method for submicrometer tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording, at different wavelengths equally separated in the k domain, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A CCD camera records the holograms consecutively, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefront. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and destructive addition in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables one to scan the object in depth. For the presented simulations and experiments, 20 wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices 725 nm thick.     

Spatial filtering in digital holographic microscopy

Abstract

An optical system based on digital holography suitable for microscopic investigations is described. A lensless digital hologram of the object under test is recorded on a CCD faceplate. The reference point source and the object are equidistant from the CCD. The point source for the illumination of the transparent microscopic object is located in another plane some millimetres behind the object. For digital reconstruction of the wavefronts the Sommerfeld propagation relation is used. The particular recording arrangement allows one to perform spatial filtering. Examples of amplitude filtering are presented.     

Dual-wavelength slightly off-axis digital holographic microscopy

We propose dual-wavelength digital holographic microscopy with a slightly off-axis configuration. The axial measurement range without phase ambiguity is extended to the micrometer range by synthesizing a beat wavelength between the two wavelengths with separation of 157 nm. Real-time measurement of the specimen is made possible by virtue of the high wavelength selectivity of the Bayer mosaic filtered color CCD camera. The principle of the method is exposed, and the practicability of the proposed configuration is demonstrated by the experimental results on a vortex phase plate and a rectangular phase step.     

Aberration compensation of holographic particle images using digital holographic microscopy

Characterisation of small and large-scale vortices in turbulent flows demands a system with high spatial resolution. The measurement of high spatial resolution, three-dimensional vector displacements in fluid mechanics using holography, is usually hampered by aberration. Aberration poses some problems in particle image identification due to low fidelity of real image reconstruction. Phase mismatch between the recording and the reconstruction waves was identified as the main source of aberration in this study. This paper demonstrates how aberration compensation can be achieved by cross-correlating the complex amplitude of an aberrated reconstructed object with the phase conjugate of a known reference object in the plane of the hologram (frequency space). Results favourably show significant increase in Strehl ratio and suppression of background noise that are more pronounced for particle images of 10 and 5 microns. It is clear from the work conducted that wavefront aberration measurement and compensation of holographic microscopic objects are now possible with the use of a variant digital holographic microscope.     

Coherence effects in digital in-line holographic microscopy

We analyze the effects of partial coherence in the image formation of a digital in-line holographic microscope (DIHM). The impulse response is described as a function of cross-spectral density of the light used in the space-frequency domain. Numerical simulation based on the applied model shows that a reduction in coherence of light leads to broadening of the impulse response. This is also validated by results from experiments wherein a DIHM is used to image latex beads using light with different spatial and temporal coherence.     

Short-coherence digital microscopy by use of a lensless holographic imaging system

An optical system based on short-coherence digital holography suitable for three-dimensional (3D) microscopic investigations is described. The light source is a short-coherence laser, and the holograms are recorded on a CCD sensor. The interference (hologram) occurs only when the path lengths of the reference and the object beam are matched within the coherence length of the laser. The image of the part of the sample that matches the reference beam is reconstructed by numerical evaluation of the hologram. The advantages of the method are high numerical aperture (this means high spatial resolution), detection of the 3D shape, and a lensless imaging system. Experimental results are presented.     

Antifouling mechanism of natural product-based coatings investigated by digital holographic microscopy

Using natural product-based antifouling coatings has proven to be an effective strategy to combat biofoul-ing.However,their antifouling mechanisms are still unclear.In this study,the antifouling mechanism of natural product-based coatings consisting of bio-sourced poly(lactic acid)-based polyurethane and eco-friendly antifoulant(butenolide)derived from marine bacteria was revealed by observing 3D bacterial motions utilizing a 3D tracking technique-digital holographic microscopy(DHM).As butenolide content increases,the density of planktonic marine bacteria(Pseudomonas sp.)near the surface decreases and thus leads to a reduced adhesion,indicating that butenolide elicits the adaptive response of Pseudomonas sp.to escape from the surface.Meanwhile,among these remained cells,an increased percentage is found to undergo subdiffusive motions compared with the case of smaller dose of butenolide.Further experiments show that butenolide can accelerate their swimming velocity and reduce flick frequency.Antibacterial assay confirms that butenolide-based coating shows high efficacy of antifouling performance against Pseudomonas sp.but without killing them like 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one(DCOIT).     

Microlens characterization by digital holographic microscopy with physical spherical phase compensation

Microlenses have been characterized by a digital holographic microscopy system, which is immune to the inherent wavefront aberration. The digital holographic microscopy system takes advantage of fiber optics and uses the light emitted directly from a single-mode fiber as the recording reference wave. By using such a reference beam, which is quasi-identical to the object beam, the inherent wavefront aberration of the digital holographic microscope is removed. The alignment of the optical setup can be optimized with the help of numerical reconstruction software to give the system phase with the off-axis tilt removed. There is one, and only one, reference fiber point position to give a reference wavefront that is quasi-identical to the object wavefront where the system is free of wavefront aberration and directly gives the quantitative phase of the test object without the need for complicated numerical compensation.     

Time-domain optical coherence tomography with digital holographic microscopy

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the short-coherence length of light source. A metallic object, covered by a 150-microm-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 microm underneath the eye surface.     

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.     

High topographical accuracy by optical shot noise reduction in digital holographic microscopy

In this work, we present a new method to reduce the shot noise in phase imaging of digital holograms. A spatial averaging process of phase images reconstructed at different reconstruction distances is performed, with the reconstruction distance range being specified by the numerical focus depth of the optical system. An improved phase image is attained with a 50% shot noise reduction. We use the integral of the angular spectrum as a reconstruction method to obtain a single-object complex amplitude that is needed to perform our proposal. We also show the corresponding simulations and experimental results. The topography of a homemade TiO2 stepwise of 100 nm high was measured and compared with the atomic force microscope results.     

Determination of the refractive index of dehydrated cells by means of digital holographic microscopy

Spatial distributions of the integral refractive index in dehydrated cells of human oral cavity epithelium are obtained by means of digital holographic microscopy, and mean refractive index of the cells is determined. The statistical analysis of the data obtained is carried out, and absolute errors of the method are estimated for different experimental conditions.     

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.     

High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy

A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.     

High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy

Abstract

A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.     

Theoretical analysis of confocal microscopy with microlenses

Scanning confocal microscopy is well established and applied frequently in biomedical science and more recently in engineering disciplines. For technical applications a confocal principle based on microlens arrays was developed. The principle permits a high depth resolution on a large field. A theoretical analysis of this principle together with some experimental results are presented.     

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.     

Measurement of microchannel flow with digital holographic microscopy by integrated nearest neighbor and cross-correlation particle pairing

A micro digital in-line holographic particle tracking velocimetry (micro-DHPTV) system has been developed and applied to investigate the three-dimensional flow field in straight and Y-junction microchannels. The micro-DHPTV system comprises a cooled frame-transfer CCD camera and a double-pulsed laser. The processing algorithm introduced to evaluate the three-dimensional velocity is based on the combination of integrated cross-correlation and nearest neighbor matching algorithms, taking advantage of information from both the reconstructed particle field and the original holograms fringes patterns. Tests on simulated pairs of holograms show that the particles can be detected, located, and paired with high probability and accuracy. Results obtained in the straight and Y-junction microchannels show that the superimposed vector field is physically reasonable.     

Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.     

Partial spatial coherence effects in digital holographic microscopy with a laser source

We investigate a digital holographic microscope that permits us to modify the spatial coherence state of the sample illumination by changing the spot size of a laser beam on a rotating ground glass. Out-of-focus planes are refocused by digital holographic reconstruction with numerical implementation of the Kirchhoff-Fresnel integral. The partial coherence nature of the illumination reduces the coherent artifact noise with respect to fully coherent illumination. The investigated configuration allows the spatial coherence state to be changed without modifying the illumination level of the sample. The effect of the coherence state on the digital holographic reconstruction is theoretically and experimentally evaluated. We also show how multiple reflection interferences are limited by the use of reduced spatial coherent illumination.