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.     

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.     

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.     

Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy

It is shown that the spatial frequencies recorded in interferometric synthetic aperture microscopy do not correspond to exact backscattering [as they do in unistatic synthetic aperture radar (SAR)] and that the reconstruction process based on SAR is therefore based on an approximation. The spatial frequency response is developed based on the three-dimensional coherent transfer function approach and compared with that in optical coherence tomography and digital holographic microscopy.     

Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence

A digital holographic technique is implemented in a microscope for three-dimensional imaging reconstruction. The setup is a Mach-Zehnder interferometer that uses an incoherent light source to remove the coherent noise that is inherent in the laser sources. A phase-stepping technique determines the optical phase in the image plane of the microscope. Out-of-focus planes are refocused by digital holographic computations, thus considerably enlarging the depth of investigation without the need to change the optical focus mechanically. The technique can be implemented in transmission for various magnification ratios and can cover a wide range of applications. Performances and limitations of the microscope are theoretically evaluated. Experimental results for a test target are given, and examples of two applications in particle localization and investigation of biological sample are provided.     

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.     

Characterization of microlenses by digital holographic microscopy

We demonstrate the use of digital holographic microscopy (DHM) as a metrological tool in micro-optics testing. Measurement principles are compared with those performed with Twyman-Green, Mach-Zehnder, and white-light interferometers. Measurements performed on refractive microlenses with reflection DHM are compared with measurements performed with standard interferometers. Key features of DHM such as digital focusing, measurement of shape differences with respect to a perfect model, surface roughness measurements, and optical performance evaluation are discussed. The capability of imaging nonspherical lenses without any modification of the optomechanical setup is a key advantage of DHM compared with conventional measurement tools and is demonstrated on a cylindrical microlens and a square lens array.     

Fourier-domain digital holographic optical coherence imaging of living tissue

Digital holographic optical coherence imaging is a full-frame coherence-gated imaging approach that uses a CCD camera to record and reconstruct digital holograms from living tissue. Recording digital holograms at the optical Fourier plane has advantages for diffuse targets compared with Fresnel off-axis digital holography. A digital hologram captured at the Fourier plane requires only a 2D fast Fourier transform for numerical reconstruction. We have applied this technique for the depth-resolved imaging of rat osteogenic tumor multicellular spheroids and acquired cross-section images of the anterior segment and the retinal region of a mouse eye. A penetration depth of 1.4 mm for the tumor spheroids was achieved.     

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.     

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.     

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.     

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.     

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.     

Multiple-wavelength digital holographic interferometry using tunable laser diodes

Here we present multiple-wavelength digital holographic interferometry with a wide measurement range using laser diodes. Small wavelength differences can be easily realized by the wavelength tuning of laser diodes with injection current controls. A contour map of an object with a wide measurement range and a high sensitivity is demonstrated by combining a few contour maps with several measurement sensitivities. Synthetic wavelengths are calibrated using a known height difference. This alleviates the need to have high precise knowledge of the recording wavelengths. The synthetic wavelengths ranged from ~3 mm for high measurement sensitivity to ~4 cm for wide measurement range. An rms error of ~35 mum for a ~1 cm height measurement is shown. The measured profile of holographic interferometry agrees with a standard stylus instrument.     

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.     

采用长工作距离物镜的低噪声相移数字全息显微研究

在数字全息显微技术中,为了提高测量精度,提出了一种利用长工作距离物镜的相移数字全息显微的测量装置和方法。该装置采用LED作为照明光源,可以有效地抑制相位噪声,提高了重建精度。通过在长工作距离物镜和样品之间加入分光棱镜的方法,构建了一种准物参共路的迈克尔逊干涉仪。该装置结构简单,调整方便,在部分相干光照明时,容易实现干涉。重建时,采用盲相移干涉技术,结合两步盲相移算法,重建出物体的表面相位分布。实验中,分别采用LED照明和He-Ne激光照明,测量了一个反射式USAF1951分辨率板的高度分布。结果表明,两者的测量结果相互吻合,但是LED照明时的噪声与激光照明时相比降低了70%。此外,为了进一步验证装置的有效性,使用该装置对刻于硅基底的微纳矩形台阶进行测量,测量结果与标称值具有良好的一致性,表明该装置在微结构的形貌测量方面有广阔的应用前景。     

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.     

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.