Recent technical advances in whole slide imaging instrumentation.

Microscopic observation of biological specimen smears is the mainstay of diagnostic pathology, as defined by the Digital Pathology Association. Though automated systems for this are commercially available, their bulky size and high cost renders them unusable for remote areas. The research community is investing much effort towards building equivalent but portable, low-cost systems. An overview of such research is presented here, including a comparative analysis of recent reports. This paper also reviews recently reported systems for automated staining and smear formation, including microfluidic devices; and optical and computational automated microscopy systems including smartphone-based devices. Image pre-processing and analysis methods for automated diagnosis are also briefly discussed. It concludes with a set of foreseeable research directions that could lead to affordable, integrated and accurate whole slide imaging systems.

Diagnosis of some diseases such as cervical cancer is done using a microscope, and this process still relies heavily on human experts. Since the need for such diagnosis is increasing at a rapid pace, it makes a lot of sense to automate the whole process. This requires automatic microscopes, which should be able to take images of a 'slide' - a glass slab with colorized human cells at its surface. These images should get analyzed by a software, resulting in a fully automated diagnosis. This article reviews recent research into this field, especially the technical advances on the hardware for automated microscopes (also known as slide imagers). It compares research reports and highlights how there's still more effort needed to build low-cost, yet clinically useful systems. It also highlights some of the emerging technologies that can be integrated into slide imagers to enable new kinds of diagnostics.

Single-shot circular fringe projection for the profiling of objects having surface discontinuities.

Fringe projection profilometry (FPP) is a widely used non-contact optical method for 3D profiling of objects. The commonly used linear fringe pattern in FPP has periodic intensity variations along the lateral direction. As a result, the linear fringe pattern used in FPP cannot uniquely represent the lateral shift induced by the objects having surface discontinuities. Thus, unambiguous surface profiling of objects, especially with surface discontinuities, using a single linear fringe image having a single fringe frequency, is unfeasible. This paper proposes using a radially symmetric circular fringe pattern as the structured light pattern for accurate unambiguous surface profiling of sudden height-discontinuous objects. To the best of our knowledge, this is the only method that can reconstruct discontinuous height profiles with the help of a single fringe image having a single frequency. The performance of the proposed algorithm is evaluated on several synthetic and real objects having smooth variations and discontinuities. Compared to the well-known fringe projection methods, the results depict that for a tolerable range of error, the proposed method can be applied for the reconstruction of objects with 4 times higher dynamic range and even at much lower fringe frequencies.

A rapid aptamer-based fluorescence assay for the detection of lipopolysaccharides using SYBR Green I.

Lipopolysaccharides (endotoxins), found on Gram-negative bacteria, can trigger a severe immune response in humans leading to septic shock and in extreme cases, even death. Therefore, the detection and neutralization of lipopolysaccharides (LPS) is of utmost importance in the pharmaceutical and medical industries. The United States Food and Drug Administration (US FDA) recommended detection method for LPS, the Limulus amebocyte lysate (LAL) assay, is expensive, time consuming, complex, and is prone to interference from proteases. As an alternative, this paper proposes a rapid, label-free fluorescence-based assay using LPS-specific aptamers and the SYBR Green DNA stain. The proposed method has a detection limit of 0.1 ng/ml, which is sufficient to detect the permissible levels of LPS in many pharmaceutical drugs and medical products. The fluorescence signal was found to be a linear function of the concentration of LPS in the range from 0.1 ng/ml to 105 ng/ml.

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

Framework for morphometric classification of cells in imaging flow cytometry.

Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high-throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi-automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph-based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost-effective microfluidics-based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost-effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis.

Handheld Fluorescence Microscopy based Flow Analyzer.

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry.

In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per µl) obtained from our instrument, with that of a commercially available hematology analyzer.

Portable optofluidic absorption flow analyzer for quantitative malaria diagnosis from whole blood.

Fast and automated diagnostic devices are bound to play a significant role in the on-going efforts toward malaria eradication. In this article, we present the realization of a portable device for quantitative malaria diagnostic testing at the point-of-care. The device measures optical absorbance (at λ=405 nm) of single cells flowing through a custom-designed microfluidic channel. The device incorporates the required functionality to align the microfluidic channel with the optical interrogation region. Variation in optical absorbance is used to differentiate red blood cells (both healthy and infected) from other cellular components of whole blood. Using the instrument, we have measured single-cell optical absorbance levels of different types of cells present in blood. High-throughput single-cell-level measurements facilitated by the device enable detection of malaria, even from a few microliters of blood. Further, we demonstrate the detection of malaria from a suspension containing all cellular components of whole blood, which validates its usability in real-world diagnostic scenarios.

Improved quantitative visualization of hypervelocity flow through wavefront estimation based on shadow casting of sinusoidal gratings.

A simple noninterferometric optical probe is developed to estimate wavefront distortion suffered by a plane wave in its passage through density variations in a hypersonic flow obstructed by a test model in a typical shock tunnel. The probe has a plane light wave trans-illuminating the flow and casting a shadow of a continuous-tone sinusoidal grating. Through a geometrical optics, eikonal approximation to the distorted wavefront, a bilinear approximation to it is related to the location-dependent shift (distortion) suffered by the grating, which can be read out space-continuously from the projected grating image. The processing of the grating shadow is done through an efficient Fourier fringe analysis scheme, either with a windowed or global Fourier transform (WFT and FT). For comparison, wavefront slopes are also estimated from shadows of random-dot patterns, processed through cross correlation. The measured slopes are suitably unwrapped by using a discrete cosine transform (DCT)-based phase unwrapping procedure, and also through iterative procedures. The unwrapped phase information is used in an iterative scheme, for a full quantitative recovery of density distribution in the shock around the model, through refraction tomographic inversion. Hypersonic flow field parameters around a missile-shaped body at a free-stream Mach number of ∼8 measured using this technique are compared with the numerically estimated values. It is shown that, while processing a wavefront with small space-bandwidth product (SBP) the FT inversion gave accurate results with computational efficiency; computation-intensive WFT was needed for similar results when dealing with larger SBP wavefronts.

Signal tracking approach for phase estimation in digital holographic interferometry.

In this research work, we introduce a novel approach for phase estimation from noisy reconstructed interference fields in digital holographic interferometry using an unscented Kalman filter. Unlike conventionally used unwrapping algorithms and piecewise polynomial approximation approaches, this paper proposes, for the first time to the best of our knowledge, a signal tracking approach for phase estimation. The state space model derived in this approach is inspired from the Taylor series expansion of the phase function as the process model, and polar to Cartesian conversion as the measurement model. We have characterized our approach by simulations and validated the performance on experimental data (holograms) recorded under various practical conditions. Our study reveals that the proposed approach, when compared with various phase estimation methods available in the literature, outperforms at lower SNR values (i.e., especially in the range 0-20 dB). It is demonstrated with experimental data as well that the proposed approach is a better choice for estimating rapidly varying phase with high dynamic range and noise.

Fluorescence imaging of flowing cells using a temporally coded excitation.

Imaging fluorescence in moving cells is fundamentally challenging because the exposure time is constrained by motion-blur, which limits the available signal. We report a method to image fluorescently labeled leukemia cells in fluid flow that has an effective exposure time of up to 50 times the motion-blur limit. Flowing cells are illuminated with a pseudo-random excitation pulse sequence, resulting in a motion-blur that can be computationally removed to produce near diffraction-limited images. This method enables observation of cellular organelles and their behavior in a fluid environment that resembles the vasculature.

Estimation of multiple phases from a single fringe pattern in digital holographic interferometry.

Simultaneous measurement of multidimensional displacements using digital holographic interferometry involves multi-directional illumination of the deformed object and requires the reliable estimation of the resulting multiple interference phase distributions. The paper introduces an elegant method to simultaneously estimate the desired multiple phases from a single fringe pattern. The proposed method relies on modeling the reconstructed interference field as a piecewise multicomponent polynomial phase signal. Effectively, in a given region or segment, the reconstructed interference field is represented as the sum of different components i.e. complex signals with polynomial phases. The corresponding polynomial coefficients are estimated using the product high-order ambiguity function. To ensure proper matching of the estimated coefficients with the corresponding components, an amplitude based discrimination criterion is used. The main advantage of the proposed method is direct retrieval of multiple phases without the application of spatial carrier based filtering operations.

Phase imaging flow cytometry using a focus-stack collecting microscope.

This letter introduces a fluidics-based focus-stack collecting microscope. A microfluidic device transports cells through the focal plane of a microscope, resulting in an efficient method to collect focus stacks of large collections of single cells. Images from the focus stacks are used to reconstruct the quantitative phase of cells with the transport-of-intensity-equation method. Using the phase imaging flow cytometer, we measure three-dimensional shape variations of red blood and leukemia cells.

Optical absorbance-based rapid test for the detection of sickle cell trait and sickle cell disease at the point-of-care.

People afflicted with sickle cell disease (SCD) experience severe deterioration in quality of life. The disease is characterized by debilitating pain, anemia, and increased susceptibility to life threatening infections. This genetic disorder is endemic to many parts of the world. Extensive and accurate screening of individuals with sickle cell trait (SCT) in the population, coupled with genetic counselling can inhibit the propagation of the disease. The gold-standard techniques for the detection of sickle hemoglobin, such as capillary electrophoresis, HPLC, and genetic testing, are prohibitively expensive and time-consuming. Mass screening is usually conducted with a low-cost test called the solubility test, which does not offer high specificity. This study proposes a game-changing single-step low-cost method for rapidly yet accurately screening and diagnosing SCD and SCT. This method relies on the hitherto unexplored differences in the optical absorbance between diseased, trait, and normal blood samples, under deoxygenated conditions. The proposed method was tested in two phases of clinical validation: a pilot study and a blind study. A total of 438 patient samples were tested using the proposed method across the two phases. The proposed method offers an average accuracy, sensitivity, and specificity of 97.6%, 96.9%, and 98.6%, respectively. The proposed test has the potential to obliviate the conventional two-step process of screening and diagnostic tests as it can be used at the point-of-care with minimal training and yet yield results reliable enough to assess disability benefit claims.

Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Estimation of dynamically varying displacement derivatives using fringe projection technique.

This paper presents a pseudo Wigner-Ville-distribution-based method in fringe projection for analyzing temporal behavior of the displacement derivative for a continuously deformed object. In the proposed method, a computer generated fringe pattern is projected on an object undergoing dynamic deformation, and the reflected intensity is recorded in the form of video, i.e., a stack of images are captured sequentially by a CCD camera. Each image represents a recorded fringe pattern at a particular time instant whose phase contains information about the instantaneous out-of-plane displacement or deformation with respect to the undeformed object, and the corresponding spatial phase derivative relates to the displacement derivative. Subsequently, pseudo Wigner-Ville distribution is used for instantaneous phase derivative estimation from the stack of images. Simulation and experimental results are presented to demonstrate the method's potential.

Simultaneous multidimensional deformation measurements using digital holographic moiré.

This paper proposes an elegant technique for the simultaneous measurement of in-plane and out-of-plane displacements of a deformed object in digital holographic interferometry. The measurement relies on simultaneously illuminating the object from multiple directions and using a single reference beam to interfere with the scattered object beams on the CCD plane. Numerical reconstruction provides the complex object wave-fields or complex amplitudes corresponding to prior and postdeformation states of the object. These complex amplitudes are used to generate the complex reconstructed interference field whose real part constitutes a moiré interference fringe pattern. Moiré fringes encode information about multiple phases which are extracted by introducing a spatial carrier in one of the object beams and subsequently using a Fourier transform operation. The information about the in-plane and out-of-plane displacements is then ascertained from the estimated multiple phases using sensitivity vectors of the optical configuration.

Simultaneous measurement of in-plane and out-of-plane displacement derivatives using dual-wavelength digital holographic interferometry.

The paper introduces a method for simultaneously measuring the in-plane and out-of-plane displacement derivatives of a deformed object in digital holographic interferometry. In the proposed method, lasers of different wavelengths are used to simultaneously illuminate the object along various directions such that a unique wavelength is used for a given direction. The holograms formed by multiple reference-object beam pairs of different wavelengths are recorded by a 3-color CCD camera with red, green, and blue channels. Each channel stores the hologram related to the corresponding wavelength and hence for the specific direction. The complex reconstructed interference field is obtained for each wavelength by numerical reconstruction and digital processing of the recorded holograms before and after deformation. Subsequently, the phase derivative is estimated for a given wavelength using two-dimensional pseudo Wigner-Ville distribution and the in-plane and out-of-plane components are obtained from the estimated phase derivatives using the sensitivity vectors of the optical configuration.

dsDNA-templated fluorescent copper nanoparticles for the detection of lipopolysaccharides.

The introduction of lipopolysaccharides (LPS) or endotoxins that originate from Gram-negative bacteria into the human blood stream induces a severe immune response that can lead to septic shock, and even death. Hence, the accurate detection of LPS is of great importance in the medical and pharmaceutical sectors. This paper proposes a novel label-free fluorescence assay for the detection of LPS utilizing aptamers and the interference synthesis of dsDNA-templated copper nanoparticles. The assay can be performed at room temperature and does not require expensive reagents. The proposed assay has a limit of detection of 0.95 ng ml-1 of LPS, and the fluorescence emission from the copper nanoparticles was found to vary linearly with the concentration of LPS over a wide range (1 to 105 ng ml-1) with R2 = 0.9877.