Off-axis Digital Holographic Microscopy Research Articles

Lossless and lossy compression of quantitative phase images of red blood cells obtained by digital holographic imaging.

In this paper, we evaluate lossless and lossy compression techniques to compress quantitative phase images of red blood cells (RBCs) obtained by an off-axis digital holographic microscopy (DHM). The RBC phase images are numerically reconstructed from their digital holograms and are stored in 16-bit unsigned integer format. In the case of lossless compression, predictive coding of JPEG lossless (JPEG-LS), JPEG2000, and JP3D are evaluated, and compression ratio (CR) and complexity (compression time) are compared against each other. It turns out that JP2k can outperform other methods by having the best CR. In the lossy case, JP2k and JP3D with different CRs are examined. Because some data is lost in a lossy way, the degradation level is measured by comparing different morphological and biochemical parameters of RBC before and after compression. Morphological parameters are volume, surface area, RBC diameter, sphericity index, and the biochemical cell parameter is mean corpuscular hemoglobin (MCH). Experimental results show that JP2k outperforms JP3D not only in terms of mean square error (MSE) when CR increases, but also in compression time in the lossy compression way. In addition, our compression results with both algorithms demonstrate that with high CR values the three-dimensional profile of RBC can be preserved and morphological and biochemical parameters can still be within the range of reported values.

Automatic full compensation of quantitative phase imaging in off-axis digital holographic microscopy.

An automatic method that fully compensates the quantitative phase measurements in off-axis digital holographic microscopy (DHM) is presented. The two main perturbations of the quantitative phase measurements in off-axis DHM are automatically removed. While the curvature phase flaw introduced by the microscope objective is avoided by the use of an optimized telecentric imaging system for the recording of the holograms, the remaining phase perturbation due to the tilt of the reference wave is removed by the automatic computation of a digital compensating reference wave. The method has been tested on both nonbiological and biological samples with and improving on the quality of the recovered phase maps.

Computational out-of-focus imaging increases the space–bandwidth product in lens-based coherent microscopy

The space–bandwidth product (SBP) of modern objective lenses is often significantly larger than the pixel count of opto-electronic image sensor chips, and, therefore, much of the information transmitted by the optical system cannot be adequately sampled or digitized. To resolve this mismatch, microscopes are in general designed to maintain the resolution of the optical system while significantly wasting the field of view (FOV) and SBP of the objective lens. We introduce a wide-field and high-resolution coherent imaging method that uses a stack of out-of-focus images to provide much better utilization of the SBP of an objective lens. We demonstrate our approach on a benchtop microscope by using a demagnification camera adapter to match the active area of the image sensor chip to the FOV of an objective lens. We show that the resulting spatial undersampling caused by capturing a large FOV can be mitigated through an iterative pixel super-resolution algorithm that uses e.g., ∼three to five slightly out-of-focus images, yielding an ∼8-fold increase in the SBP of the microscope. Furthermore, the same pixel super-resolution algorithm also achieves phase retrieval, revealing the optical phase information of the specimen. We compared our method against traditional off-axis and phase-shifting digital holographic microscopy modalities and demonstrated at least 3-fold reduction in the number of images required to achieve the same SBP. This technique could be used to maximize the throughput and SBP of lens-based coherent imaging and holography systems and inspire new microscopy designs that benefit from the inherent autofocusing steps of a scanning microscope to increase its SBP.

Automated Fourier space region-recognition filtering for off-axis digital holographic microscopy.

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding method to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here paves way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures.

Cell morphology-based classification of red blood cells using holographic imaging informatics.

We present methods that automatically select a linear or nonlinear classifier for red blood cell (RBC) classification by analyzing the equality of the covariance matrices in Gabor-filtered holographic images. First, the phase images of the RBCs are numerically reconstructed from their holograms, which are recorded using off-axis digital holographic microscopy (DHM). Second, each RBC is segmented using a marker-controlled watershed transform algorithm and the inner part of the RBC is identified and analyzed. Third, the Gabor wavelet transform is applied to the segmented cells to extract a series of features, which then undergo a multivariate statistical test to evaluate the equality of the covariance matrices of the different shapes of the RBCs using selected features. When these covariance matrices are not equal, a nonlinear classification scheme based on quadratic functions is applied; otherwise, a linear classification is applied. We used the stomatocyte, discocyte, and echinocyte RBC for classifier training and testing. Simulation results demonstrated that 10 of the 14 RBC features are useful in RBC classification. Experimental results also revealed that the covariance matrices of the three main RBC groups are not equal and that a nonlinear classification method has a much lower misclassification rate. The proposed automated RBC classification method has the potential for use in drug testing and the diagnosis of RBC-related diseases.

Robust, compact implementation of an off-axis digital holographic microscope.

Recent advances in digital technologies, such as high-speed computers and large-format digital imagers, have led to a burgeoning interest in the science and engineering of digital holographic microscopy (DHM). Here we report on a novel off-axis DHM, based on a twin-beam optical design, which avoids the limitations of prior systems, and provides many advantages, including compactness, intrinsic stability, robustness against misalignment, ease of use, and cost. These advantages are traded for a physically constrained sample volume, as well as a fixed fringe spacing. The first trade is not overly restrictive for most applications, and the latter provides for a pre-set assembly alignment that optimizes the spatial frequency sampling. Moreover, our new design supports use in both routine laboratory settings as well as extreme environments without any sacrifice in performance, enabling ready observation of microbial species in the field. The instrument design is presented in detail here, along with a demonstration of bacterial video imaging at sub-micrometer resolution at temperatures down to -15 °C.

In situ study of two interacting femtosecond filaments in sapphire.

Interaction dynamics of two copropagating femtosecond light filaments in sapphire crystal is studied by means of time-resolved off-axis digital holographic and shadowgraphic microscopy with 22 fs temporal and 1 μm spatial resolution. In particular, we demonstrate that filament interaction originates from instantaneous modification of the refractive index resulting from the Kerr effect. Fusion, splitting, and even suppression of the resulting plasma channels induced by interacting filaments was observed by varying time delay between the input pulses. Free electron channels were reconstructed in the form of the time-lapse movie with unprecedented spatial and temporal resolution.

Study of spatial lateral resolution in off-axis digital holographic microscopy

The lateral resolution in digital holographic microscopy (DHM) has been widely studied in terms of both recording and reconstruction parameters. Although it is understood that once the digital hologram is recorded the physical resolution is fixed according to the diffraction theory and the pixel density, still some researches link the resolution of the reconstructed wavefield with the recording distance as well as with the zero-padding technique. Aiming to help avoiding these misconceptions, in this paper we analyze the lateral resolution of DHM through the variation of those two parameters. To support our outcomes, we have designed numerical simulations and experimental verifications. Both the simulations and the experiments confirm that DHM is indeed resolution invariant in terms of the recording distance and the zero-padding provided that it operates within the angular spectrum regime.

Optimisation of a Holographic Microscope for Microbial Study


 
 
 
 Background: Tracking of microbial organisms over a volume requires images at multiple focal planes along the orthogonal direction. With most conventional microscopes, this requires repeated readjustments; using Digital Holographic Microscopy (DHM), it is possible to use a set of interference patterns to reconstruct at various distances, thereby creating a 3D stack based off a single image.
 Methods: We used an off-axis Mach-Zehnder DHM for imaging and tracking bacterial movement. We describe the algorithm employed for tracking, as well as our improvement of trackability by testing differences in image contrast with the use of Quantum Dots.
 Results: We show that the use of Quantum Dots resulted in an increase in contrast of approximately 11%.
 Conclusion: We suggest this as a method of increasing resolvability of individual microbes. With a more compact design, the microscope will be applicable in various fields, and can be used remotely for studies of microbial organisms.
 
 
 

An experimental investigation of three-dimensional particle aggregation using digital holographic microscopy

The tendency of particles to aggregate depends on particle-particle and particle-fluid interactions. These interactions can be characterized but it requires accurate 3D measurements of particle distributions. We introduce the application of an off-axis digital holographic microscopy for measuring distributions of dense micrometer (2μm) particles in a liquid solution. We demonstrate that digital holographic microscopy is capable of recording the instantaneous 3D position of particles in a flow volume. A new reconstruction method that aids identification of particle images was used in this work. About 62% of the expected number of particles within the interrogated flow volume was detected. Based on the 3D position of individual particles, the tendency of particle to aggregate is investigated. Results show that relatively few particles (around 5–10 of a cohort of 1500) were aggregates. This number did not change significantly with time.

Three-dimensional counting of morphologically normal human red blood cells via digital holographic microscopy.

Counting morphologically normal cells in human red blood cells (RBCs) is extremely beneficial in the health care field. We propose a three-dimensional (3-D) classification method of automatically determining the morphologically normal RBCs in the phase image of multiple human RBCs that are obtained by off-axis digital holographic microscopy (DHM). The RBC holograms are first recorded by DHM, and then the phase images of multiple RBCs are reconstructed by a computational numerical algorithm. To design the classifier, the three typical RBC shapes, which are stomatocyte, discocyte, and echinocyte, are used for training and testing. Nonmain or abnormal RBC shapes different from the three normal shapes are defined as the fourth category. Ten features, including projected surface area, average phase value, mean corpuscular hemoglobin, perimeter, mean corpuscular hemoglobin surface density, circularity, mean phase of center part, sphericity coefficient, elongation, and pallor, are extracted from each RBC after segmenting the reconstructed phase images by using a watershed transform algorithm. Moreover, four additional properties, such as projected surface area, perimeter, average phase value, and elongation, are measured from the inner part of each cell, which can give significant information beyond the previous 10 features for the separation of the RBC groups; these are verified in the experiment by the statistical method of Hotelling's T-quare test. We also apply the principal component analysis algorithm to reduce the dimension number of variables and establish the Gaussian mixture densities using the projected data with the first eight principal components. Consequently, the Gaussian mixtures are used to design the discriminant functions based on Bayesian decision theory. To improve the performance of the Bayes classifier and the accuracy of estimation of its error rate, the leaving-one-out technique is applied. Experimental results show that the proposed method can yield good results for calculating the percentage of each typical normal RBC shape in a reconstructed phase image of multiple RBCs that will be favorable to the analysis of RBC-related diseases. In addition, we show that the discrimination performance for the counting of normal shapes of RBCs can be improved by using 3-D features of an RBC.

Imaging‐in‐Flow: Digital holographic microscopy as a novel tool to detect and classify nanoplanktonic organisms

Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time‐ consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on “imaging‐ in‐ flow,” i.e., the ability to acquire microscopic images of planktonic cells in a flow‐ through mode. Imaging‐ in‐ flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e.g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging‐ in‐ flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off‐ axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape‐ based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic‐ based imaging‐ in‐ flow for similar looking organisms in the nanoplankton range.

An off-axis digital holographic microscope with quasimonochromatic partially spatially coherent illumination in transmission

We propose an off-axis imaging approach for digital holographic microscopy (DHM) with quasimonochromatic partially spatially coherent illumination in transmission, which is intended to provide the advantages of off-axis partially coherent DHM imaging with a comparatively simple optical scheme. This approach does not require a diffraction grating for creating the off-axis modality and also allows for convenient control of the spatial frequency of carrier interference fringes for hologram sampling optimization. Theoretical analysis of the off-axis imaging process in this microscope is performed. An off-axis DHM based on the proposed approach is built and quantitative phase imaging of test objects is performed with suppressed coherent noise.

Digital holographic microscopy with coupled optical fiber trap for cell measurement and manipulation

We present an integrated optical system for three-dimensional (3D) imaging of micrometer-sized samples, while immobilizing and manipulating the samples by means of an optical fiber trap. Optical traps allow us to apply and measure pico-Newton-sized forces, and perform detailed measurements of micrometer-sized dielectric systems in the field of biology. The integrated 3D system can be used as a major tool in the field of biophysics. The trap is built using a tapered optical fiber to enhance the effective numerical aperture of the fiber. The trapping system is mounted on a conventional microscope, in which the two eyepieces' output ports are used as the paths of an off-axis self-referencing digital holographic microscopy (DHM) setup. The trap is calibrated using a high-speed camera, and trap stiffness is determined through the power spectrum method. The compact setup provides an elegant apparatus for temporally stable DHM for 3D imaging of optically controlled samples. Three-dimensional information and quantitative phase contrast images of the trapped samples are obtained by postprocessing the recorded digital holograms. Experiments were performed on lipids and red blood cells. Quantitative phase contrast images and temporal evolution of optical thickness of trapped samples are presented.

Off-axis digital holographic microscopy: practical design parameters for operating at diffraction limit.

The utilization of microscope objectives (MOs) in digital holographic microscopy (DHM) has associated effects that are not present in conventional optical microscopy. The remaining phase curvature, which can ruin the quantitative phase imaging, is the most evident and analyzed. As phase imaging is considered, this interest has made possible the development of different methods of overcoming its undesired consequences. Additionally to the effects in phase imaging, there exist a set of less obvious conditions that have to be accounted for as MOs are utilized in DHM to achieve diffraction-limit operation. These conditions have to be considered even in the case in which only amplitude or intensity imaging is of interest. In this paper, a thorough analysis of the physical parameters that control the appropriate utilization of MOs in DHM is presented. A regular DHM system is theoretically modeled on the basis of the imaging theory. The Fourier spectrum of the recorded hologram is analyzed to evaluate the performance of the DHM. A set of the criteria that consider the microscope features and the recording parameters to achieve DHM operation at the diffraction limit is derived. Numerical modeling and experimental results are shown to validate our findings.

Automated quantitative analysis of 3D morphology and mean corpuscular hemoglobin in human red blood cells stored in different periods

Quantitative phase (QP) images of red blood cells (RBCs), which are obtained by off-axis digital holographic microscopy, can provide quantitative information about three-dimensional (3D) morphology of human RBCs and the characteristic properties such as mean corpuscular hemoglobin (MCH) and MCH surface density (MCHSD). In this paper, we investigate modifications of the 3D morphology and MCH in RBCs induced by the period of storage time for the purpose of classification of RBCs with different periods of storage by using off-axis digital holographic microscopy. The classification of RBCs based on the duration of storage is highly relevant because a long storage of blood before transfusion may alter the functionality of RBCs and, therefore, cause complications in patients. To analyze any changes in the 3D morphology and MCH of RBCs due to storage, we use data sets from RBC samples stored for 8, 13, 16, 23, 27, 30, 34, 37, 40, 47, and 57 days, respectively. The data sets consist of more than 3,300 blood cells in eleven classes, with more than 300 blood cells per class. The classes indicate the storage period of RBCs and are listed in chronological order. Using the RBCs donated by healthy persons, the off-axis digital holographic microscopy reconstructs several quantitative phase images of RBC samples stored for eleven different periods. We employ marker-controlled watershed transform to remove the background in the RBC quantitative phase images obtained by the off-axis digital holographic microscopy. More than 300 single RBCs are extracted from the segmented quantitative phase images for each class. Such a large number of RBC samples enable us to obtain statistical distributions of the characteristic properties of RBCs after a specific period of storage. Experimental results show that the 3D morphology of the RBCs, in contrast to MCH, is essentially related to the aging of the RBCs.

Off-axis digital holographic microscopy with LED illumination based on polarization filtering

A reflection mode digital holographic microscope with light emitting diode (LED) illumination and off-axis interferometry is proposed. The setup is comprised of a Linnik interferometer and a grating-based 4f imaging unit. Both object and reference waves travel coaxially and are split into multiple diffraction orders in the Fourier plane by the grating. The zeroth and first orders are filtered by a polarizing array to select orthogonally polarized object waves and reference waves. Subsequently, the object and reference waves are combined again in the output plane of the 4f system, and then the hologram with uniform contrast over the entire field of view can be acquired with the aid of a polarizer. The one-shot nature in the off-axis configuration enables an interferometric recording time on a millisecond scale. The validity of the proposed setup is illustrated by imaging nanostructured substrates, and the experimental results demonstrate that the phase noise is reduced drastically by an order of 68% when compared to a He-Ne laser-based result.

A comparison of digital holographic microscopy and on-axis phase-shifting interferometry for surface profiling

This paper presents a quantitative comparison between off-axis digital holographic microscopy (DHM) and on-axis phase-shifting interferometry (PSI) for surface micro topography measurement. The comparison has been applied on an object of a 1.34μm nominal step height. The experimental results show that single shot, dual-wavelength, off-axis DHM surpasses on dual-wavelength, on-axis PSI in terms of accuracy and repeatability.

Automated segmentation of multiple red blood cells with digital holographic microscopy

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

数字全息显微像平面相位畸变的自动补偿研究

The automatic phase aberration compensation of off-axis digital holographic microscopy is studied theoreticaly and experimentaly.Firstly,the average gradient auto-focus function is used to achieve the optimal reconstruction distance of the digital hologram recorded by CCD.After numerical apodization and spatial frequency filtering,the numerical reconstruction is performed by the product of the hologram with modified digital reference wave.Finally,with the automatic phase aberration compensation of image plane,the accurate phase distribution is achieved.This method uses only one single digital hologram and can retrieve automatically and rapidly the real phase distribution of the object.