White Light Diffraction Phase Microscopy Research Articles

White light diffraction phase microscopy for imaging of red blood cells for different storage times

In this study, the effects of different storage times on the surface morphology of red blood cells (RBCs) were investigated using white light diffraction phase microscopy (WDPM). Blood samples collected from 10 volunteer and stored for 56 days, were imaged on WDPM at every 7 days without any sample preparation. To obtain the phase profiles of RCBs, first the sample and then the reference interferograms were obtained from the experimental setup. Then, surface profiles were calculated from these interferogram images using Fourier transform (FT). With the experiment performed every 7 days, 10 RBC phase information were obtained from each sample and surface profiles were created. From these profiles, 7 parameters related with RBC morphology (average cell thickness—ACT; mean corpuscular volume—MCV; projected surface area—PSA; total surface area—SA; diameter—D; mean corpuscular haemoglobin—MCH; surface area to volume ratio - SAV) have been calculated. Therefore, changes in the morphology of RBCs during storage were evaluated quantitatively. Additionally, phase calibration target was used to confirm the accuracy of our experimental setup system. From the interferogram images, the depth of the phase target (GRP 9 and element 3) was obtained as 309 nm, in accordance with the produced depth. By this way, the reliability of the WDPM setup is demonstrated. This study suggests that the morphology of RBCs can be quantitatively obtained in a reliable manner at higher resolution with WDPM without sample preparation.

Multi-derivative method for phase extraction without knowing carrier frequencies in off-axis quantitative phase imaging.

We propose a multi-derivative method to reconstruct the phase of transparent objects in off-axis quantitative phase imaging (QPI). By numerically computing first-, second-, and third-order derivatives of the interferogram, we demonstrate that one can extract the quantitative phase information in a straightforward way, without prior knowledge of the carrier frequencies or Fourier transform. In contrast to existing advanced derivative methods, our approach markedly streamlines the alignment and retrieval processes, all without requiring any special prerequisites. This enhancement seamlessly translates into improved reconstruction quality. Furthermore, when compared to cutting-edge Fourier-division-based methods, our technique distinctly accelerates the phase retrieval speed. We verified our method using white-light diffraction phase microscopy and laser off-axis QPI, and the results indicate that our method can allow a fast, high-quality retrieval with frame rates up to 41.6 fps for one- megapixel interferograms on a regular computer.

Novel Approach to Investigate the Effect of High-Dose Methylprednisolone on Erythrocyte Morphology: White Light Diffraction Microscopy

Abstract The present study focuses on quantitative phase imaging of erythrocytes with the aim to evaluate the effects of high-dose methylprednisolone (HDMP) on erythrocytes in vivo under physiological conditions in human blood samples. Samples from ten patients, prescribed to be treated with 1000 mg/day intravenous methylprednisolone for 5 days, were analyzed by white light diffraction phase microscopy (WDPM) for quantitative imaging. WDPM, an optical measurement technique, enables single shot measurement and low speckle noise using white light. Quantitative phase imaging performed with this experimental setup allowed the determination of erythrocyte morphology with 9 different parameters. In vivo quantitative analysis of erythrocytes by WDPM, which is a simple and reliable method, shows that HDMP treatment has no significant effect on erythrocyte morphology. With the developing technology, interdisciplinary studies on individuals under treatment should play an important role in elucidating the interaction between steroids and erythrocytes.

Non-Invasive and Minute-Frequency 3D Tomographic Imaging Enabling Long-Term Spatiotemporal Observation of Single Cell Fate.

Non-invasive and rapid imaging technique at subcellular resolution is significantly important for multiple biological applications such as cell fate study. Label-free refractive-index (RI)-based 3D tomographic imaging constitutes an excellent candidate for 3D imaging of cellular structures, but its full potential in long-term spatiotemporal cell fate observation is locked due to the lack of an efficient integrated system. Here, a long-term 3D RI imaging system incorporating a cutting-edge white light diffraction phase microscopy module with spatiotemporal stability, and an acoustofluidic device to roll and culture single cells in a customized live cell culture chamber is reported. Using this system, 3D RI imaging experiments are conducted for 250 cells and demonstrate efficient cell identification with high accuracy. Importantly, long-term and frequency-on-demand 3D RI imaging of K562 and MCF-7 cancer cells reveal different characteristics during normal cell growth, drug-induced cell apoptosis, and necrosis of drug-treated cells. Overall, it is believed that the proposed 3D tomographic imaging technique opens up a new avenue for visualizing intracellular structures and will find many applications such as disease diagnosis and nanomedicine.

Quantitative Determination of Surface Morphology of Red Blood Cell

In this study, the determination of the surface morphology of red blood cell (RBC) from interferogram image obtained by quantitative phase imaging (QPI) method is presented. QPI, is an optical measurement method fre-quently used in recent years, allows to obtain quantitative data for different samples (cell, thin film surface, etc.). Many measurement setups at the micrometer level and with nanometer precision have been designed for quantita-tive surface determination. Among these, white light diffraction phase microscopy (WDPM) is a design that combines the advantages of off-axis holography-specific speed and phase sensitivity associated with common path interferometry. Interferogram image of RBCs have been formed by the WDPM setup. Analysis of this image has been carried out by Fourier transform. As a result of this analysis, three-dimensional (3D), dynamic (observable from all angles) and height-known profiles of RBCs have been created. From the height profiles, the parameters related to the morphology of RBCs as the projected surface area (PSA), diameter (D), mean corpuscular volume (MCV) and total surface area occupied by the cell (SA), have been determined quantitatively. In addition, two-dimensional images, obtained by examining blood samples with light microscopy and scanning electron micros-copy (SEM), have been compared with the data achieved by WDPM. The advantages and disadvantages of WDPM and light microscopy and SEM, which are commonly used in biomedical measurements, are discussed through the results. In this way, it was possible to see the difference between QPI and traditional methods used to imaging the cell surface.

Quantitative phase imaging of erythrocyte in epilepsy patients.

The present study focuses on the quantitative phase imaging of erythrocytes with the aim to compare the morphological differences between epilepsy patients under antiepileptic treatment, who have no other disease which may affect the erythrocyte morphology, and the healthy control group. The white light diffraction phase microscopy (WDPM) has been used to obtain the interferogram of the erythrocyte surfaces. The continuous wavelet transform with Paul wavelet has been chosen to calculate the surface profiles from this interferogram image. For the determination of alteration in morphology, besides WDPM, erythrocyte surfaces have been investigated by light microscope and scanning electron microscope. In this way, it has been possible to see the difference in terms of precision and implementation between the most commonly used methods with regard to the quantitative phase imaging. Erythrocytes from all the samples have been examined and displayed in both two- and three-dimensional way. We have observed that erythrocytes of patients with effective antiepileptic blood levels were more affected in morphology than healthy subjects. When we compared the erythrocyte morphological changes of patients who received monotherapy or polytherapy, no difference was observed. In conclusion, antiepileptic drugs (AEDs) cause red blood cell (RBC) morphological changes and a combined usage of WDPM with Paul wavelet and light microscopy methods are very convenient for studying the erythrocyte morphologies on multiple patients.

白光衍射相位像光晕效应消除方法

白光衍射相位像光晕效应消除方法

White light diffraction phase microscopy with slightly off-axis blind two-step phase-shifting

White light diffraction phase microscopy (wDPM) combines many of the best attributes of current quantitative phase imaging (QPI) techniques, which fully takes advantages of both high temporal and spatial phase sensitivity granted by the common-path geometry and the white light illumination. However, like all off-axis quantitative phase imaging methods, although wDPM benefits from the acquisition rate due to the single shot measurement, it is characterized by a reduced space–bandwidth product, compared to phase shifting approaches. In the paper, we develop slightly off-axis blind two-step phase-shifting wDPM, measuring two phase-shifted interferograms by blindly shifting the grating. Due to the combination of phase-shifting and off-axis techniques, the time–bandwidth and space–bandwidth products are well traded off. In addition, the phase-shifting is simply implemented by blindly shifting the grating with a stepping motor linear translation stage, not requiring a precise phase modulator. Thus, the apparatus is cost-effective and offers ease of alignment. Some experimental results are given to demonstrate the feasibility of the method.

White-light diffraction phase microscopy at doubled space-bandwidth product.

White light diffraction microscopy (wDPM) is a quantitative phase imaging method that benefits from both temporal and spatial phase sensitivity, granted, respectively, by the common-path geometry and white light illumination. However, like all off-axis quantitative phase imaging methods, wDPM is characterized by a reduced space-bandwidth product compared to phase shifting approaches. This happens essentially because the ultimate resolution of the image is governed by the period of the interferogram and not just the diffraction limit. As a result, off-axis techniques generates single-shot, i.e., high time-bandwidth, phase measurements, at the expense of either spatial resolution or field of view. Here, we show that combining phase-shifting and off-axis, the original space-bandwidth is preserved. Specifically, we developed phase-shifting diffraction phase microscopy with white light, in which we measure and combine two phase shifted interferograms. Due to the white light illumination, the phase images are characterized by low spatial noise, i.e., <1nm pathlength. We illustrate the operation of the instrument with test samples, blood cells, and unlabeled prostate tissue biopsy.

WHITE LIGHT DIFFRACTION PHASE MICROSCOPY

Quantitative phase imaging (QPI) techniques are very advantageous compared to the qualitative phase imaging techniques like bright field microscopy, phase contrast microscopy, differential interference contrast microscopy due to measurement of phase information quantitatively. In QPI techniques, there is no need of exogenous contrast agents to stain or tag the specimen. White light diffraction phase microscopy (wDPM) is one of the QPI techniques which is used to quantify the phase information from the samples. The phase information retrieved is dependent on the local refractive index and thickness of the biological cells. So, by measuring the phase one can find the refractive index and/or thickness of the specimen in nanometer scales. In this technique, the acquisition speed is only limited by the sensing device due to its single shot feature, it is better compared to other quantitative phase imaging techniques as its common path geometry provides better temporal phase sensitivity by cancelling out the most mechanisms responsible for noise and with the use of white light source the images are speckle-free. So we achieve better spatial phase sensitivity compared to other techniques. This method has attractive applications in biomedicine. We have developed wDPM set up for extracting phase information from the live yeast cells and sperm cells. The phase is reconstructed from the recorded interferograms by using a fast phase reconstruction algorithm. This algorithm avoids the unwrapping step which is needed in conventional phase reconstruction by Hilbert’s transform method. We have calculated the thickness of the yeast cells and sperm cells and demonstrate a video for visualizing the dynamics of yeast cells.

White light diffraction phase microscopy as profilometry tool

A reflection type white light diffraction phase microscope for full field surface profiling of opaque samples is proposed. The system can extract surface profile from one recorded interferogram without any mechanical movement and the use of white light makes it free from speckle noise. We validated the performance of our system by measuring a known step sample and a high-quality plane sample. The step height of the step sample is found to be 88.5 nm with a standard deviation of 1.4 nm, and the surface peak to valley value of the plane sample is found to be 28.6 nm with a standard deviation of 3 nm.

Effects of spatial coherence in diffraction phase microscopy

Quantitative phase imaging systems using white light illumination can exhibit lower noise figures than laser-based systems. However, they can also suffer from object-dependent artifacts, such as halos, which prevent accurate reconstruction of the surface topography. In this work, we show that white light diffraction phase microscopy using a standard halogen lamp can produce accurate height maps of even the most challenging structures provided that there is proper spatial filtering at: 1) the condenser to ensure adequate spatial coherence and 2) the output Fourier plane to produce a uniform reference beam. We explain that these object-dependent artifacts are a high-pass filtering phenomenon, establish design guidelines to reduce the artifacts, and then apply these guidelines to eliminate the halo effect. Since a spatially incoherent source requires significant spatial filtering, the irradiance is lower and proportionally longer exposure times are needed. To circumvent this tradeoff, we demonstrate that a supercontinuum laser, due to its high radiance, can provide accurate measurements with reduced exposure times, allowing for fast dynamic measurements.

Fast phase reconstruction in white light diffraction phase microscopy

In off-axis interferometry, we usually have to deal with the unwrapping process, which is very computationally intensive and prevents us from real time phase reconstruction. The wrapping problem usually occurs when imaging thick objects, which introduce phase shifts of more than 2π radians. However, in off-axis interferometry, the nonzero angle of interference of the two beams creates a ramp in the phase across the image that can produce phase wrapping errors. In this paper, we propose a simple technique that avoids the need for the unwrapping step in reconstructing quantitative phase images in white light diffraction phase microscopy of thin samples. We show that this approach can improve significantly the phase reconstruction speed and allow high impact applications, such as real-time blood testing.

Diffraction phase microscopy with white light

We present white light diffraction phase microscopy (wDPM) as a quantitative phase imaging method that combines the single shot measurement benefit associated with off-axis methods, high temporal phase stability associated with common path geometries, and high spatial phase sensitivity due to the white light illumination. We propose a spatiotemporal filtering method that pushes the limit of the pathlength sensitivity to the subangstrom level at practical spatial and temporal bandwidths. We illustrate the utility of wDPM with measurements on red blood cell morphology and HeLa cell growth over 18 hours.