Dual-wavelength Digital Holographic Microscopy Research Articles

Simultaneous dual-wavelength digital holographic microscopy as a tool for the analysis of keratoacanthoma skin samples

A keratoacanthoma (KA) skin tumor is usually caused by sun exposure and may be an alert sign prior to the development of a more aggressive tumor or skin cancer. Studying the shape of the KA cells and their 3D rendering visualization are important parameters to prevent its evolution to higher stages of tumor cells or skin cancer. KA cells shape can be obtained through digital holographic microscopy; for that purpose, a setup with two illumination wavelengths (532 and 638 nm) is implemented to render a synthetic wavelength of 3.2 μm that avoids wrapping the optical phase of the processed holograms and increases measurement range. To recover the optical phase, two off-axis digital holograms are simultaneously recorded at each wavelength. From the processed hologram height variations, the shape and length of KA cells, as well as the stratum corneum epidermal layer, are obtained as phase images. The results achieved aid to discriminate healthy from malignant cells.

Phase compensation algorithm based on image segmentation in dual-wavelength holographic microscopy.

In order to solve the problem of phase compensation errors in the traditional 2π phase compensation method caused by a rough surface and complex structure of objects in dual-wavelength digital holographic microscopy, a phase compensation algorithm based on image segmentation was proposed. First, the phase less than zero in the phase obtained by an equivalent wavelength is compensated for by adding 2π initially. Then the phase after the initial compensation is binarized, and the small connected areas in the binarized graph are removed, so as to obtain a new binarized graph. Finally, according to the two binarized graphs, the phase of the object after the initial 2π phase compensation is recompensated for in different regions, so as to obtain the continuous phase distribution of the object. Based on the dual-wavelength digital holographic microscopy experimental system with an adjustable equivalent wavelength, the proposed algorithm is used to perform three-dimensional imaging of the channel of the microfluidic chip. The experimental results show that the proposed method can effectively obtain the continuous real phase of the object when the structure of the object is known, so as to obtain a more accurate and reliable three-dimensional topography of the object. The above results provide a new idea for the high-quality three-dimensional imaging of the microfluidic system.

The phase range extension and accuracy improvement in Fresnel biprism-based digital holography microscopy

We report a highly stable and affordable dual-wavelength digital holographic microscopy system based on common-path geometry. A Fresnel biprism is used to create an off-axis geometry, and two diode laser sources with different wavelengths λ1 = 532nm and λ2 = 650nm generate the dual-wavelength compound hologram. In order to extend the measurement range, the phase distribution is obtained using a synthetic wavelength Λ1 = 2930.5nm. Furthermore, to improve the system's temporal stability and reduce speckle noise, a shorter wavelength (Λ2 = 292.5nm) is used. The feasibility of the proposed configuration is validated by the experimental results obtained with Molybdenum trioxide, Paramecium, and red blood cell specimens.

Single-Shot Common-Path Off-Axis Dual-Wavelength Digital Holographic Microscopy Based on Two-Dimensional Grating Diffraction

We present a single-shot dual-wavelength common-path off-axis digital holographic microscopic (CO-DHM) imaging method based on two-dimensional grating diffraction. This method improves the utilization rate of the interference field under the limited photosensitive size of the camera, and further expands the original camera’s field of view (FOV). In addition, the mode of orthogonal carrier frequencies close to the diagonal direction can optimize the utilization of the camera’s spatial bandwidth. Compared with the traditional dual-wavelength CO-DHM using one-dimensional grating or prism beam splitting, this method effectively avoids the aliasing of high-frequency components of the +1-order spectrum of different wavelengths in the frequency domain. We provide quantitative phase imaging experiments for the full FOV of USAF resolution chart, onion epidermal cells and standard polystyrene beads. The results prove that the system can enlarge the interferometric FOV by nearly 74.0% without changing the imaging parameters, such as magnification and resolution, and can achieve high-precision quantitative phase imaging with only a single hologram.

Robust and fast dual-wavelength phase unwrapping in quantitative phase imaging with region segmentation

Dual-wavelength digital holographic microscopy extends the unambiguous measurement range in quantitative phase imaging. However, it suffers from increased noise, which reduces phase sensitivity, especially when a larger beat wavelength is synthesized. We present a simple, robust, and fast dual-wavelength phase unwrapping method, without increased phase noise, by segmenting a single wavelength phase map into multiple regions along its phase discontinuities and calculating the fringe order of each region. The method works properly with a large beat wavelength, while reducing phase noise to the level of a single wavelength. Simulations were performed for imaging a smooth profile sample and a multi-heights step. We also demonstrated its practical implementation in quantitative imaging of microstructures where a beat wavelength was up to 20.475μm.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Autofocusing based on cosine similarity in dual-wavelength digital holographic microscopy

An autofocusing method based on cosine similarity in dual-wavelength digital holographic microscopy is proposed. In our experiments, red and green lasers are employed for illumination and the generated holograms are recorded by a color camera. During the refocusing process, the reconstructed optical field near the focus plane contains more regular features than the one on the defocus plane. Moreover, due to the wavelength dependency of the diffraction process, the further away from the focus plane, the greater the difference in the reconstructed optical fields from the two wavelengths. Therefore, the focus plane can be determined by finding the maximum value of the cosine similarity between the amplitude vectors from the reconstructed optical fields of the two wavelengths. Simulation and experimental results demonstrate the effectiveness of the proposed method in off-axis dual-wavelength digital holographic microscopy for shape measurement.

Single-shot common-path off-axis dual-wavelength digital holographic microscopy

A single-shot common-path off-axis self-interference dual-wavelength digital holographic microscopic (DHM) system based on a cube beam splitter is demonstrated to expand the phase range in a stepped microstructure and for simultaneous measurement of the refractive index and physical thickness of a specimen. In the system, two laser beams with wavelengths of 532 nm and 632.8 nm are used. These laser beams are combined to transilluminate the object under study, then the object beam is divided into two beams by using a beam splitter oriented in such a way that both the beams propagate in almost the same direction, with an appropriate lateral separation between them. One of the object beams is spatially filtered at its Fourier plane, using a pinhole to generate a reference spherical beam free from the object information. The reference beam interferes with the object beam to form a digital hologram at the faceplate of the image sensor. The phase information is extracted from a single recorded digital hologram using the phase aberration compensation method that is based on principal component analysis (PCA). Owing to the common-path configuration, the system shows high temporal phase stability and it is less vibration-sensitive compared to counterparts such as a Mach-Zehnder type DHM. The performance of the dual-wavelength DHM system is verified in two different application fields by conducting the experiments using microsphere beads and living plant cells.

Hierarchical phase unwrapping for dual-wavelength digital holographic microscopy

Dual-wavelength digital holographic microscopy (DHM) can extend ambiguity range in phase unwrapping by synthesizing a much longer beat wavelength, but it suffers from phase error amplification that destroys the vertical sensitivity and accuracy. To achieve a better vertical sensitivity and avoid height jump phenomenon, a hierarchical method is presented to construct a wavelength chain and unwrap the phase gradually from long to short wavelengths. This method could improve the vertical sensitivity by synthesizing a shorter wavelength and avoid height jump by reducing the estimation error of integer part of phase order. Theoretical analysis, simulations, and experiments demonstrate the effectiveness of the hierarchical method to attenuate the height jump and preserve vertical sensitivity in dual-wavelength DHM phase unwrapping. The method is simple and deterministic, and only costs a few milliseconds for unwrapping a typical phase map of mega pixels.

Simultaneous dual-wavelength digital holographic microscopy with compensation of chromatic aberration for accurate surface characterization.

In this paper, a simultaneous dual-wavelength digital holographic microscope with compensation of chromatic aberration is presented. The microscope has been used to calibrate two standard step heights of 1340 and 1000 nm. The extracted results have been compared with those extracted by the single-shot, dual-wavelength digital holographic microscope, and the comparison has shown that measurements by the proposed microscope are more accurate by around 40 nm. We claim that the high accuracy in measurement is due to the fact that the proposed system works in real time with very low chromatic aberration. Judging the performance of the measurement process over time is explained by the control charts. Evidence from the control measurements sets a probable limit on precision for this process at about 3σ=3.5 nm.

Dual-wavelength digital holographic phase and fluorescence microscopy combining with Raman spectroscopy for micro-quartz pieces-based dual-channel encoded suspension array.

Dual-wavelength digital holographic phase and fluorescence microscopy (DW-DHPFM), combining with Raman spectroscopy, is designed to achieve the detection and analysis of biomolecules with a new dual-channel encoding method. This employs the Raman reporter molecules assembled micro-quartz pieces (MQPs) as microcarriers of suspension array (SA). The dual-wavelength digital holographic phase microscopy (DW-DHPM) and Raman spectroscopy are served as the decoding platforms, and the fluorescence microscopy is used to quantify target analytes. Considering the independence between encoding and label signal, the above two encoding channels could effectively avoid the crosstalk in immunoassay process, and the combination of two encoding methods expand the encoding capacity with a considerable magnitude. Accurate and stable decoding abilities are verified by multiplexed immunoassay experiment and the quantitative analysis of targets with high-sensitivity is confirmed by concentration gradient experiments.

Study on Quantitative Phase Imaging by Dual-Wavelength Digital Holography Microscopy

A dual-wavelength digital holographic microscopy with premagnification is proposed to obtain the object surface measurements over the large gradient. The quantitative phase images of specimens are captured in high precision by the processing of filtering and phase compensation. The phase images are acquired without phase unwrapping, which is necessary in traditional digital holographic microscopy; thereby the proposed system can greatly increase the speed of reconstruction. The results of numerical simulation and optical experiments demonstrated that the reconstructed speed increased by 37.9 times, and the relative error of measurement is 4% compared with the traditional holographic microscopy system. It means that the proposed system can directly acquire the higher quality quantitative phase distribution for specimens.

A Method to Improve the Imaging Quality in Dual-Wavelength Digital Holographic Microscopy.

A digital hologram-optimizing method was proposed to improve the imaging quality of dual-wavelength digital holographic microscopy (DDHM) by reducing the phase noise level. In our previous work, phase noise reduction was achieved by dual-wavelength digital image-plane holographic microscopy (DDIPHM). In the optimization method in this paper, the phase noise was further reduced by enhancing the real-image term and suppressing effects of the zero-order term in the frequency spectrum of a digital hologram. Practically, the carrier frequency of the real-image term has the correspondence with interference fringes in the hologram. Mathematically, the first order intrinsic mode function (IMF1) in bidimensional empirical mode decomposition (BEMD) has similar characteristics to the grayscale values of ideal interference fringes. Therefore, with the combination of DDIPHM and BEMD, by utilizing the characteristics of IMF1, the digital hologram was optimized with purified interference fringes, enhancing the real-image term simultaneously. Finally, the validity of the proposed method was verified by experimental results on a microstep.

Phase unwrapping in dual-wavelength digital holographic microscopy with total variation regularization.

A two-step dual-wavelength phase imaging method with total variation (TV) regularization in digital holographic microscopy (DHM) is presented. The rough height map is first denoised with TV regularization, and then it is used as a guiding map for accurate phase unwrapping. By combining the principle of dual-wavelength interferometry and TV regularization, the problem of phase unwrapping is converted into a minimization problem that can be efficiently solved with the Split Bregman algorithm. The proposed algorithm allows a synthetic wavelength that can be arbitrarily large in principle to a single wavelength, while the accuracy of the phase map can achieve the same level in single-wavelength DHM. Moreover, the unwrapping method is robust to the change of weighting parameters. Comparative topographic measurements of a sample with abrupt steps are illustrated, and the results verified the effectiveness of the method.

Compact and stable real-time dual-wavelength digital holographic microscopy with a long-working distance objective.

We propose a compact dual wavelength digital holographic Microscopy (DHM) based on a long working distance objective, which enabling quantitative phase imaging of opaque samples with extended measurement range in one shot. The compactness of the configuration is achieved by constructing a miniature modified Michelson interferometer between the objective and the sample, and as a result it provides higher temporal stability than conventional dual wavelength DHM. In the setup, the propagation directions of two reference beams of different wavelengths can be independently adjusted, and thus two off axis interferograms having orthogonal fringe directions can be simultaneously captured through a monochrome CCD camera. The unambiguous vertical measurement range in optical path length is extended to 8.338 μm, the length of a synthetic wavelength, by selecting two wavelengths with a gap of 52 nm. The capability of the proposed setup is demonstrated with measurements of a standard 1.8 μm height step as well as a moving micro staircase structure.

Single-shot dual-wavelength phase reconstruction in off-axis digital holography with polarization-multiplexing transmission.

A new system for single-shot dual-wavelength digital holographic microscopy with polarization-multiplexing path-shared transmission is presented. The key feature of the optical configuration is that the interference waves of two wavelengths having orthogonal polarization can transmit in the same interferometer paths at the same time, and two polarizers orthogonal to each other are placed in front of the CCD to realize single-shot recording of two holograms. The correlative filtering algorithm of the spatial-frequency spectrum for dual-wavelength digital holograms is reliable and efficient in the dual-wavelength path-shared configuration. The phase reconstruction in dual-wavelength digital holographic imaging is achieved by using this filtering algorithm. The experiment results of phase reconstruction of a groove grating demonstrate the reliability and validity of this optical configuration and the correlative filtering algorithm. This polarization-multiplexing configuration for dual-wavelength digital holography is compact and has more flexibility for the replacement of different-wavelength lasers.

Improvement of measurement accuracy in digital holographic microscopy by using dual-wavelength technique

In traditional dual-wavelength digital holographic microscopy (DHM), a synthetic wavelength is obtained by using two lasers with different wavelengths, and the measurement range of the samples’ step height can be expanded from nanometers to micrometers. However, the measurement accuracy reduces along with the expansion of the measuring range, and significant noise is simultaneously introduced in this process. For cases where the sample’s step height is smaller than the wavelength of the illumination light, the measurement accuracy is very important. We present a new approach for dual-wavelength DHM. The synthetic wavelength is shorter than either of the two lasers, and thus higher measurement accuracy can be achieved. The numerical simulation and experiment results show the feasibility of this technique.

Determining the refractive index distribution of optical components by dual-wavelength digital holographic microscopy with a liquid

A method is proposed for measuring the two-dimensional distribution of the refractive index in transparent micro-optical components. The proposed method combines dual-wavelength digital holographic microscopy with liquids to record holograms at different wavelengths and to numerically reconstruct the three-dimensional phase information to obtain the sample’s distribution of the refractive index. A theoretical model is proposed for determining the two-dimensional distribution of the refractive index; an achromatic lens and plate are demonstrated experimentally. Unlike conventional scanning interferometry, the proposed method supports non-scanning measurement and direct observation, independent of the sample’s shape, of the two-dimensional distribution of refractive indices in micro-optical elements.

基于彩色CMOS双波长数字全息显微术的细胞相位定量测量

基于彩色CMOS双波长数字全息显微术的细胞相位定量测量

Digital holographic microscope employing dual-Lloyd’s mirror

We propose a digital holographic microscopy (DHM) setup employing a configuration with two Lloyd's mirrors, which is based on self-referencing and dual-wavelength optical phase unwrapping. We use two Lloyd's mirrors to fold the beam which does not exhibit sample structure and acts as the reference beam, returning onto itself to form a dual-wavelength hologram. Two wrapped phase images for every wavelength are reconstructed by angular spectrum method. Then the wrapped phase image and the three-dimensional profile image are acquired by dual-wavelength optical unwrapping method. In the experiment, we use two lasers of different wavelengths of 532 and 632 nm to record a hologram. Numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. The quantitative experimental results with dual-wavelength DHM involve a deviation less than 5% from the calibration values. The validity of this method is demonstrated.