Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells.

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

Automated tilt compensation in acoustic microscopy.

Scanning acoustic microscopy (SAM) is a potent and nondestructive technique capable of producing three-dimensional topographic and tomographic images of specimens. This is achieved by measuring the differences in time of flight (ToF) of acoustic signals emitted from various regions of the sample. The measurement accuracy of SAM strongly depends on the ToF measurement, which is affected by tilt in either the scanning stage or the sample stage. Hence, compensating for the ToF shift resulting from sample tilt is imperative for obtaining precise topographic and tomographic profiles of the samples in a SAM. In the present work, we propose an automated tilt compensation in ToF of acoustic signal based on proposed curve fitting method. Unlike the conventional method, the proposed approach does not demand manually choosing three separate coordinate points from SAM's time domain data. The effectiveness of the proposed curve fitting method is demonstrated by compensating time shifts in ToF data of a coin due to the presence of tilt. The method is implemented for the correction of different amounts of tilt in the coin corresponding to angles 6.67°, 12.65° and 15.95°. It is observed that the present method can perform time offset correction in the time domain data of SAM with an accuracy of 45 arcsec. The experimental results confirm the effectiveness of the suggested tilt compensation technique in SAM, indicating its potential for future applications.

Laser-Generated Scholte Waves in Floating Microparticles.

This study aims to demonstrate the generation and detection of Scholte waves inside polystyrene microparticles. This was proven using both experimental analysis and COMSOL simulation. Microspheres of different sizes were excited optically with a pulsed laser (532 nm), and the acoustic signals were detected using a transducer (40 MHz). On analyzing the laser-generated ultrasound signals, the results obtained experimentally and from COMSOL are in close agreement both in the time and frequency domain. A simplified analysis of Scholte wave generation by laser irradiation for homogeneous, isotropic microspheres is presented. The theoretical wave velocity of the Scholte wave was calculated and found close to our experimental results. A representation of pressure wave motion showing the Scholte wave generation is presented at different times.

Effects of Saccharomyces cerevisiae live cells and culture on growth and productive performance in lactating Nili-Ravi buffaloes.

An experimental work was conducted to evaluate the effects of Saccharomyces cerevisiae live cells and its culture on dry matter intake (DMI), milk yield, milk composition, body condition score, selected blood metabolites, feed conversion efficiency (FCE), nutrient digestibility, body weight gain, and economics of milk production in lactating multiparous Nili-Ravi buffaloes. In total, 20 buffaloes of age 5 years ± 6 months and weighing 550 ± 20 kg were selected and assigned to four dietary treatments (n=5 buffalo/treatment) under completely randomized design. The dietary treatments include treatment 1 (T1) control, treatment 2 (T2) 5g/head live yeast, treatment 3 (T3) 5g/head yeast culture, and treatment 4 (T4) 10 g/head yeast culture per day for 60 days excluding 14 days as an adjustment period. The results indicated that T4 showed significant (p<0.05) improvement in DMI, milk yield and components, blood glucose level, digestibility of nutrients, and body weight gain while significant decrease in blood urea nitrogen as compared to other treatment groups. Body condition score was not affected among treatments. In conclusion, yeast culture supplementation significantly improved (p <0.05) milk yield, milk composition, DMI, body weight gain, blood glucose level, and digestibility while significantly decreased blood urea level as compare to control. Economic return was also improved. BCS was not improved. Comparatively, yeast culture showed significant improvement in growth and productive performance as compare to live yeast. Meanwhile, 10-g yeast culture showed better results as compare to 5-g yeast culture.

Hilbert phase microscopy based on pseudo thermal illumination in the Linnik configuration.

Quantitative phase microscopy (QPM) is often based on recording an object-reference interference pattern and its further phase demodulation. We propose pseudo Hilbert phase microscopy (PHPM) where we combine pseudo thermal light source illumination and Hilbert spiral transform (HST) phase demodulation to achieve hybrid hardware-software-driven noise robustness and an increase in resolution of single-shot coherent QPM. Those advantageous features stem from physically altering the laser spatial coherence and numerically restoring spectrally overlapped object spatial frequencies. The capabilities of PHPM are demonstrated by analyzing calibrated phase targets and live HeLa cells in comparison with laser illumination and phase demodulation via temporal phase shifting (TPS) and Fourier transform (FT) techniques. The performed studies verified the unique ability of PHPM to combine single-shot imaging, noise minimization, and preservation of phase details.

Multiple Damage Detection in PZT Sensor Using Dual Point Contact Method.

Lead Zirconate Titanate (PZT) is used to make ultrasound transducers, sensors, and actuators due to its large piezoelectric coefficient. Several micro-defects develop in the PZT sensor due to delamination, corrosion, huge temperature fluctuation, etc., causing a decline in its performance. It is thus necessary to identify, locate, and quantify the defects. Non-Destructive Structural Health Monitoring (SHM) is the most optimal and economical evaluation method. Traditional ultrasound SHM techniques have a huge impedance mismatch between air and solid material, and most of the popular signal processing methods define time series signals in only one domain, which provides sub-optimal results for non-stationary signals. Thus, to improve the accuracy of detection, the point contact excitation and detection method is implemented to determine the interaction of ultrasonic waves with micro-scale defects in the PZT. The signal generated from this method being non-stationary in nature, it requires signal processing with changeable resolutions at different times and frequencies. The Haar Discrete Wavelet Transformation (DWT) is applied to the time series data obtained from the coulomb coupling setup. Using the above process, defects up to 100 µm in diameter could be successfully distinguished.

Single-shot fringe pattern phase retrieval using improved period-guided bidimensional empirical mode decomposition and Hilbert transform.

Fringe pattern analysis is the central aspect of numerous optical measurement methods, e.g., interferometry, fringe projection, digital holography, quantitative phase microscopy. Experimental fringe patterns always contain significant features originating from fluctuating environment, optical system and illumination quality, and the sample itself that severely affect analysis outcome. Before the stage of phase retrieval (information decoding) interferogram needs proper filtering, which minimizes the impact of mentioned issues. In this paper we propose fully automatic and adaptive fringe pattern pre-processing technique - improved period guided bidimensional empirical mode decomposition algorithm (iPGBEMD). It is based on our previous work about PGBEMD which eliminated the mode-mixing phenomenon and made the empirical mode decomposition fully adaptive. In present work we overcame key problems of original PGBEMD - we have considerably increased algorithm's application range and shortened computation time several-fold. We proposed three solutions to the problem of erroneous decomposition for very low fringe amplitude images, which limited original PGBEMD significantly and we have chosen the best one among them after comprehensive analysis. Several acceleration methods were also proposed and merged to ensure the best results. We combined our improved pre-processing algorithm with the Hilbert Spiral Transform to receive complete, consistent, and versatile fringe pattern analysis path. Quality and effectiveness evaluation, in comparison with selected reference methods, is provided using numerical simulations and experimental fringe data.

Sampling moiré method: a tool for sensing quadratic phase distortion and its correction for accurate quantitative phase microscopy.

The advantages of quantitative phase microscopy (QPM) such as label-free imaging with high spatial sensitivity, live cell compatibility and high-speed imaging makes it viable for various biological applications. The measurement accuracy of QPM strongly relies on the shape of the recorded interferograms, whether straight or curved fringes are recorded during the data acquisition. Moreover, for a single shot phase recovery high fringe density is required. The wavefront curvature for the high-density fringes over the entire field of view is difficult to be discerned with the naked eye. As a consequence, there is a quadratic phase aberration in the recovered phase images due to curvature mismatch. In the present work, we have implemented sampling moiré method for real-time sensing of the wavefront curvature mismatch between the object and the reference wavefronts and further for its correction. By zooming out the interferogram, moiré fringes are generated which helps to easily identify the curvature of the fringes. The wavefront curvature mismatch correction accuracy of the method is tested with the help of low temporal coherent light source such as a white light (temporal coherence ∼ 1.6 µm). The proposed scheme is successfully demonstrated to remove the quadratic phase aberration caused due to wavefront mismatch from an USAF resolution target and the biological tissue samples. The phase recovery accuracy of the current scheme is further compared with and found to better than the standard method called principle component analysis. The proposed method enables recording of the corrected wavefront interferogram without needing any additional optical components or modification and also does not need any post-processing correction algorithms. The proposed method of curvature compensation paves the path for a high-throughput and accurate quantitative phase imaging.

Sub-nanometer height sensitivity by phase shifting interference microscopy under environmental fluctuations.

Phase shifting interferometric (PSI) techniques are among the most sensitive phase measurement methods. Owing to its high sensitivity, any minute phase change caused due to environmental instability results into, inaccurate phase measurement. Consequently, a well calibrated piezo electric transducer (PZT) and highly-stable environment is mandatory for measuring accurate phase map using PSI implementation. Here, we present an inverse approach, which can retrieve phase maps of the samples with negligible errors under environmental fluctuations. The method is implemented by recording a video of continuous temporally phase shifted interferograms and phase shifts were calculated between all the data frames using Fourier transform algorithm with a high accuracy ≤ 5.5 × 10-4 π rad. To demonstrate the robustness of the proposed method, a manual translation of the stage was employed to introduce continuous temporal phase shift between data frames. The developed algorithm is first verified by performing quantitative phase imaging of optical waveguide and red blood cells using uncalibrated PZT under the influence of vibrations/air turbulence and compared with the well calibrated PZT results. Furthermore, we demonstrated the potential of the proposed approach by acquiring the quantitative phase imaging of an optical waveguide with a rib height of only 2 nm and liver sinusoidal endothelial cells (LSECs). By using 12-bit CMOS camera the height of shallow rib waveguide is measured with a height sensitivity of 4 Å without using PZT and in presence of environmental fluctuations.vn.

Automatic fringe pattern enhancement using truly adaptive period-guided bidimensional empirical mode decomposition.

Fringe patterns encode the information about the result of a measurement performed via widely used optical full-field testing methods, e.g., interferometry, digital holographic microscopy, moiré techniques, structured illumination etc. Affected by the optical setup, changing environment and the sample itself fringe patterns are often corrupted with substantial noise, strong and uneven background illumination and exhibit low contrast. Fringe pattern enhancement, i.e., noise minimization and background term removal, at the pre-processing stage prior to the phase map calculation (for the measurement result decoding) is therefore essential to minimize the jeopardizing effect the mentioned error sources have on the optical measurement outcome. In this contribution we propose an automatic, robust and highly effective fringe pattern enhancement method based on the novel period-guided bidimensional empirical mode decomposition algorithm (PG-BEMD). The spatial distribution of the fringe period is estimated using the novel windowed approach and then serves as an indicator for the truly adaptive decomposition with the filter size locally adjusted to the fringe pattern density. In this way the fringe term is successfully extracted in a single (first) decomposition component alleviating the cumbersome mode mixing phenomenon and greatly simplifying the automatic signal reconstruction. Hence, the fringe term is dissected without the need for modes selection nor summation. The noise removal robustness is ensured employing the block matching 3D filtering of the fringe pattern prior to its decomposition. Performance validation against previously reported modified empirical mode decomposition techniques is provided using numerical simulations and experimental data verifying the versatility and effectiveness of the proposed approach.

High space-bandwidth in quantitative phase imaging using partially spatially coherent digital holographic microscopy and a deep neural network.

Quantitative phase microscopy (QPM) is a label-free technique that enables monitoring of morphological changes at the subcellular level. The performance of the QPM system in terms of spatial sensitivity and resolution depends on the coherence properties of the light source and the numerical aperture (NA) of objective lenses. Here, we propose high space-bandwidth quantitative phase imaging using partially spatially coherent digital holographic microscopy (PSC-DHM) assisted with a deep neural network. The PSC source synthesized to improve the spatial sensitivity of the reconstructed phase map from the interferometric images. Further, compatible generative adversarial network (GAN) is used and trained with paired low-resolution (LR) and high-resolution (HR) datasets acquired from the PSC-DHM system. The training of the network is performed on two different types of samples, i.e. mostly homogenous human red blood cells (RBC), and on highly heterogeneous macrophages. The performance is evaluated by predicting the HR images from the datasets captured with a low NA lens and compared with the actual HR phase images. An improvement of 9× in the space-bandwidth product is demonstrated for both RBC and macrophages datasets. We believe that the PSC-DHM + GAN approach would be applicable in single-shot label free tissue imaging, disease classification and other high-resolution tomography applications by utilizing the longitudinal spatial coherence properties of the light source.

Characterization of color cross-talk of CCD detectors and its influence in multispectral quantitative phase imaging.

Multi-spectral quantitative phase imaging (QPI) is an emerging imaging modality for wavelength dependent studies of several biological and industrial specimens. Simultaneous multi-spectral QPI is generally performed with color CCD cameras. Here, we present a new approach for accurately measuring the color crosstalk of 2D area detectors, without needing prior information about camera specifications. Color crosstalk is systematically studied and compared using compact interference microscopy on two different cameras commonly used in QPI, single chip CCD (1-CCD) and three chip CCD (3-CCD). The influence of color crosstalk on the fringe width and the visibility of the monochromatic constituents corresponding to three color channels of white light interferogram are studied both through simulations and experiments. It is observed that presence of color crosstalk changes the fringe width and visibility over the imaging field of view. This leads to an unwanted non-uniform background error in the multi-spectral phase imaging of the specimens. The color crosstalk of the detector is observed to be the limiting factor for phase measurement accuracy of simultaneous multi-spectral QPI systems.

Effect on the longitudinal coherence properties of a pseudothermal light source as a function of source size and temporal coherence.

In the present Letter, a synthesized pseudothermal light source having high temporal coherence (TC) and low spatial coherence (SC) properties is used. The longitudinal coherence (LC) properties of the spatially extended monochromatic light source are systematically studied. The pseudothermal light source is generated from two different monochromatic laser sources: He-Ne (at 632 nm) and DPSS (at 532 nm). It was found that the LC length of such a light source becomes independent of the parent laser's TC length for a large source size. For the chosen lasers, the LC length becomes constant to about 30 µm for a laser source size of ≥3.3 mm. Thus, by appropriately choosing the source size, any monochromatic laser light source depending on the biological window can be utilized to obtain high axial resolution in an optical coherence tomography (OCT) system irrespective of its TC length. The axial resolution of 650 nm was obtained using a 1.2 numerical aperture objective lens at a 632 nm wavelength. These findings pave the path for widespread penetration of pseudothermal light into existing OCT systems with enhanced performance. A pseudothermal light source with high TC and low SC properties could be an attractive alternative light source for achieving high axial resolution without needing dispersion compensation as compared to a broadband light source.

Relationship between the source size at the diffuser plane and the longitudinal spatial coherence function of the optical coherence microscopy system.

Coherence properties of light sources are indispensable for optical coherence microscopy/tomography as they greatly influence the signal-to-noise ratio, axial resolution, and penetration depth of the system. In the present paper, we report the investigation of longitudinal spatial coherence properties of a pseudothermal light source (PTS) as a function of the laser spot size at the rotating diffuser plate. The laser spot size is varied by translating a microscope objective lens toward or away from the diffuser plate. The longitudinal spatial coherence length, which governs the axial resolution of the coherence microscope, is found to be minimum for the beam spot size of 3.5 mm at the diffuser plate. The axial resolution of the system is found to be equal to an $\sim{13}\,\,{\rm \unicode{x00B5}{\rm m}}$∼13µm at 3.5 mm beam spot size. The change in the axial resolution of the system is confirmed by performing the experiments on standard gauge blocks of a height difference of 15 µm by varying the spot size at the diffuser plate. Thus, by appropriately choosing the beam spot size at the diffuser plane, any monochromatic laser light source can be utilized to obtain high axial resolution irrespective of the source's temporal coherence length. It can provide speckle-free tomographic images of multilayered biological specimens with large penetration depth. In addition, a PTS avoids the use of any chromatic-aberration-corrected optics and dispersion-compensation mechanism unlike conventional setups.

Low coherence quantitative phase microscopy with machine learning model and Raman spectroscopy for the study of breast cancer cells and their classification.

Early-stage detection of breast cancer is the primary requirement in modern healthcare as it is the most common cancer among women worldwide. Histopathology is the most widely preferred method for the diagnosis of breast cancer, but it requires long processing time and involves qualitative assessment of cancer by a trained person/doctor. Here, we present an alternate technique based on white light interference microscopy (WLIM) and Raman spectroscopy, which has the capability to differentiate between cancerous and normal breast tissue. WLIM provides quantitative phase information about the biological tissues/cells, whereas Raman spectroscopy can detect changes in their molecular structure and chemical composition during cancer growth. Further, both the techniques can be implemented very quickly without staining the sample. The present technique is employed to perform ex vivo study on a total of 80 normal and cancerous tissue samples collected from 16 different patients. A generalized machine learning model is developed for the classification of normal and cancerous tissues, which is based on texture features obtained from phase maps with an accuracy of 90.6%. The correlation of outcomes from these two techniques can open a new avenue for fast and accurate detection of cancer without any trained personnel.

Volumetric analysis of breast cancer tissues using machine learning and swept-source optical coherence tomography.

In breast cancer, 20%-30% of cases require a second surgery because of incomplete excision of malignant tissues. Therefore, to avoid the risk of recurrence, accurate detection of the cancer margin by the clinician or surgeons needs some assistance. In this paper, an automated volumetric analysis of normal and breast cancer tissue is done by a machine learning algorithm to separate them into two classes. The proposed method is based on a support-vector-machine-based classifier by dissociating 10 features extracted from the A-line, texture, and phase map by the swept-source optical coherence tomographic intensity and phase images. A set of 88 freshly excised breast tissue [44 normal and 44 cancers (invasive ductal carcinoma tissues)] samples from 22 patients was used in our study. The algorithm successfully classifies the cancerous tissue with sensitivity, specificity, and accuracy of 91.56%, 93.86%, and 92.71% respectively. The present computational technique is fast, simple, and sensitive, and extracts features from the whole volume of the tissue, which does not require any special tissue preparation nor an expert to analyze the breast cancer as required in histopathology. Diagnosis of breast cancer by extracting quantitative features from optical coherence tomographic images could be a potentially powerful method for cancer detection and would be a valuable tool for a fine-needle-guided biopsy.

Multi-modal chip-based fluorescence and quantitative phase microscopy for studying inflammation in macrophages.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Quantitative phase imaging of biological cells using spatially low and temporally high coherent light source.

In this Letter, we demonstrate quantitative phase imaging of biological samples, such as human red blood cells (RBCs) and onion cells using narrow temporal frequency and wide angular frequency spectrum light source. This type of light source was synthesized by the combined effect of spatial, angular, and temporal diversity of speckle reduction technique. The importance of using low spatial and high temporal coherence light source over the broad band and narrow band light source is that it does not require any dispersion compensation mechanism for biological samples. Further, it avoids the formation of speckle or spurious fringes which arises while using narrow band light source.

Multispectral quantitative phase imaging of human red blood cells using inexpensive narrowband multicolor LEDs.

We report multispectral phase-shifting interference microscopy for quantitative phase imaging of human red blood cells (RBCs). A wide range of wavelengths are covered by means of using multiple color light emitting diodes (LEDs) with narrow spectral bandwidth ranging from violet to deep red color. The multicolor LED light source was designed and operated sequentially, which works as a multispectral scanning light source. Corresponding to each color LED source, five phase-shifted interferograms were recorded sequentially for the measurement of phase maps, as well as the refractive index of RBCs within the entire visible region. The proposed technique provides information about the effect of wavelengths on the morphology and refractive index of human RBCs. The system does not require expensive multiple color filters or any wavelength scanning mechanism along with broadband light source.

High space-time bandwidth product imaging in low coherence quantitative phase microscopy.

Current low coherence quantitative phase microscopy (LC-QPM) systems suffer from either reduced field of view (FoV) or reduced temporal resolution due to the short temporal coherence (TC) length of the light source. Here, we propose a hybrid, experimental and numerical approach to address this core problem associated with LC-QPM. We demonstrate high spatial resolution and high phase sensitivity in LC-QPM at high temporal resolution. High space-time bandwidth product is achieved by employing incoherent light source for sample illumination in QPM to increase the spatial resolution and single-shot Hilbert spiral transform (HST) based phase recovery algorithm to enhance the temporal resolution without sacrificing spatial resolution during the reconstruction steps. The high spatial phase sensitivity comes by default due to the use of incoherent light source in QPM which has low temporal coherence length and does not generate speckle noise and coherent noise. The spatial resolution achieved by the HST is slightly inferior to the temporal phase-shifting (TPS) method when tested on a specimen but surpasses that of the single-shot Fourier transform (FT) based phase recovery method. Contrary to HST method, FT method requires high density fringes for lossless phase recovery, which is difficult to achieve in LC-QPM over entire FoV. Consequently, integration of HST algorithm with LC-QPM system makes an attractive route. Here, we demonstrate scalable FoV and resolution in single-shot LC-QPM and experimentally corroborate it on a test object and on both live and fixed biological specimen such as MEF, U2OS and human red blood cells (RBCs). LC-QPM system with HST reconstruction offer high-speed single-shot QPM imaging at high phase sensitivity and high spatial resolution enabling us to study sub-cellular dynamic inside U2OS for extended duration (3 h) and observe high-speed (50 fps) dynamics of human RBCs. The experimental results validate the effectiveness of the present approach and will open new avenues in the domain of biomedical imaging in the future.