arXiv subjects

Vishesh Dubey

Publications and source records attributed to Vishesh Dubey.

10 recordsLinked to original sources

arXiv2026

Lateral shearing optical diffraction tomography of brain organoid with reduced spatial coherence

Optical diffraction tomography (ODT) is a powerful technique for quantitative, label-free reconstruction of the three-dimensional refractive index (RI) distribution of biological samples. While ODT is well established for imaging thin, weakly scattering samples, it encounters significant challenges when applied to heterogeneous, strongly scattering thick samples such as tissues and organoids. In this work, a novel common-path interferometric approach to ODT is presented, specifically designed for the RI reconstruction of heterogeneous and highly scattering samples at high temporal stability. The proposed technique, termed lateral shearing (LS)-ODT, incorporates partial lateral shearing off-axis interferometry to suppress the effects of multiple scattering, similar to the mechanism in differential interference contrast (DIC) microscopy, which is widely used for imaging thick specimens. Additionally, the LS-ODT system uses dynamic speckle illumination to enhance both spatial phase and RI sensitivity compared to laser-based ODT systems. The effectiveness of this method is demonstrated through experiments on a cell phantom. Its robustness and accuracy are further validated across a wide range of samples, including mouse kidney tissue sections and brain organoids derived from human induced pluripotent stem cells (iPSCs), in both thin and thick sections. Furthermore, correlative fluorescence and RI tomography of the organoids highlights the potential of LS-ODT to enhance and support a broad spectrum of biomedical studies, particularly in the field of histology.

physics.optics↗

arXiv2022

Label-free chip-based evanescent light scattering super-resolution and superior-contrast optical microscopy (cELS)

Chip-based Evanescent Light Scattering (cELS) utilizes the multiple modes of a high-index contrast optical waveguide for near-field illumination of unlabeled samples, thereby repositioning the highest spatial frequencies of the sample into the far-field. The multiple modes scattering off the sample with different phase differences is engineered to have random spatial distributions within the integration time of the camera, mitigating the coherent speckle noise. This enables label-free superior-contrast imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs) and liposomes, dynamics of living HeLa cells etc. The article explains and validates experimentally the physics behind cELS by demonstrating a multi-moded straight waveguide as a partially coherent light source. For isotropic super-resolution, spatially incoherent light engineered via multiple-arms waveguide chip and intensity-fluctuation based algorithms are used. The proof-of-concept results are demonstrated on 100 nm polystyrene beads and resolution improvement of close to 2X is shown. cELS also realizes (2-10)X more contrast as opposed to conventional imaging techniques. In addition, cELS platform is miniaturized and enables large field-of-view imaging compared to state of the art label-free techniques. cELS holds a potential for label-free super-resolution imaging of nanosized biological specimens at high-throughput.

physics.optics↗

arXiv2020

High throughput spatially sensitive single-shot quantitative phase microscopy

High space-bandwidth product with high spatial phase sensitivity is indispensable for a single-shot quantitative phase microscopy (QPM) system. It opens avenue for widespread applications of QPM in the field of biomedical imaging. Temporally low coherence length light sources are generally implemented to achieve high spatial phase sensitivity in QPM at the cost of either reduced temporal resolution or smaller field of view (FOV). On the contrary, high temporal coherence light sources like lasers are capable of exploiting the full FOV of the QPM systems at the expense of less spatial phase sensitivity. In the present work, we employed pseudo-thermal light source (PTLS) in QPM which overcomes the limitations of conventional light sources. The capabilities of PTLS over conventional light sources are systematically studied and demonstrated on various test objects like USAF resolution chart and thin optical waveguide (height ~ 8 nm). The spatial phase sensitivity of QPM in case of PTLS is measured to be equivalent to that for white light source. The high-speed and large FOV capabilities of PTLS based QPM is demonstrated by high-speed imaging of live sperm cells that is limited by the camera speed and by imaging extra-ordinary large FOV phase imaging on histopathology placenta tissue samples.

physics.optics↗

arXiv2020

Deep learning architecture LightOCT for diagnostic decision support using optical coherence tomography images of biological samples

Optical coherence tomography (OCT) is being increasingly adopted as a label-free and non-invasive technique for biomedical applications such as cancer and ocular disease diagnosis. Diagnostic information for these tissues is manifest in textural and geometric features of the OCT images, which are used by human expertise to interpret and triage. However, it suffers delays due to the long process of the conventional diagnostic procedure and shortage of human expertise. Here, a custom deep learning architecture, LightOCT, is proposed for the classification of OCT images into diagnostically relevant classes. LightOCT is a convolutional neural network with only two convolutional layers and a fully connected layer, but it is shown to provide excellent training and test results for diverse OCT image datasets. We show that LightOCT provides 98.9% accuracy in classifying 44 normal and 44 malignant (invasive ductal carcinoma) breast tissue volumetric OCT images. Also, >96% accuracy in classifying public datasets of ocular OCT images as normal, age-related macular degeneration and diabetic macular edema. Additionally, we show ~96% test accuracy for classifying retinal images as belonging to choroidal neovascularization, diabetic macular edema, drusen, and normal samples on a large public dataset of more than 100,000 images. The performance of the architecture is compared with transfer learning based deep neural networks. Through this, we show that LightOCT can provide significant diagnostic support for a variety of OCT images with sufficient training and minimal hyper-parameter tuning. The trained LightOCT networks for the three-classification problem will be released online to support transfer learning on other datasets.

physics.med-ph↗

arXiv2020

A transparent waveguide chip for versatile TIRF-based microscopy and nanoscopy

Total internal reflection fluorescence microscopy (TIRF) has enabled low-background, live-cell friendly imaging of cell surfaces and other thin samples thanks to the shallow penetration of the evanescent light field into the sample. The implementation of TIRF on optical waveguide chips (c-TIRF) has overcome historical limitations on the magnification and field of view (FOV) compared to lens-based TIRF, and further allows the light to be guided in complicated patterns that can be used for advanced imaging techniques or selective stimulation of the sample. However, the opacity of the chips themselves has thus far precluded their use on inverted microscopes and complicated sample preparation and handling. In this work, we introduce a new platform for c-TIRF imaging based on a transparent substrate, which is fully compatible with sample handling and imaging procedures commonly used with a standard #1.5 glass coverslip, and is fabricated using standard complementary metal-oxide-semiconductor (CMOS) techniques, which can easily be scaled up for mass production. We demonstrate its performance on synthetic and biological samples using both upright and inverted microscopes, and show how it can be extended to super-resolution applications, achieving a resolution of 116 nm using super resolution radial fluctuations (SRRF). These new chips retain the scalable FOV of opaque chip-based TIRF and the high axial resolution of TIRF, and have the versatility to be used with many different objective lenses, microscopy methods, and handling techniques. We thus see c-TIRF as a technology primed for widespread adoption, increasing both TIRF's accessibility to users and the range of applications that can benefit from it.

physics.app-ph↗

arXiv2019

Influence of laser spot size at diffuser plane on the longitudinal spatial coherence function of optical coherence microscopy system

Coherence properties and wavelength 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 letter, we investigated the longitudinal spatial coherence properties of the pseudo-thermal light source (PTS) as a function of spot size at the diffuser plane, which is controlled by translating microscope objective lens towards or away from the diffuser plane. The axial resolution of PTS is found to be maximum ~ 13 microns for the beam spot size of 3.5 mm at the diffuser plane. The change in the axial resolution of the system as the spot size is increased at the diffuser plane is further confirmed by performing experiments on standard gauge blocks of height difference of 15 microns. Thus, by appropriately choosing the beam spot size at the diffuser plane, any monochromatic laser light source depending on the biological window can be utilized to obtain high axial-resolution with large penetration depth and speckle-free tomographic images of multilayered biological specimens irrespective of the source temporal coherence length. In addition, PTS could be an attractive alternative light source for achieving high axial-resolution without needing chromatic aberration corrected optics and dispersion-compensation mechanism, unlike conventional setups.

physics.optics↗

arXiv2018

Highly stable common-path quantitative phase microscope for biomedical imaging

High temporal stability is the primary requirement of any quantitative phase microscope (QPM) systems for the early stage detection of various human related diseases. The high temporal stability of the system provides accurate measurement of membrane fluctuations of the biological cells, which can be good indicator of various diseases. We developed a single element highly stable common-path QPM system to obtain temporally stable holograms of the biological specimens. With the proposed system, the temporal stability is obtained ~ 15 mrad without using any vibration isolation table. The capability of the proposed system is demonstrated on USAF resolution chart, polystyrene spheres (dia. 4.5 micron) and human red blood cells (RBCs). The membrane fluctuation of healthy human RBCs is further successfully measured and found to be equal to 63 nm. Contrary to its counterparts, present system offers energy efficient, cost effective and simple way of generating object and reference beam for the development of common-path QPM.

physics.optics↗

arXiv2018

Sub-nanometer height sensitivity by phase shifting interference microscopy under ambient 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 a new method of recording temporal phase shifted interferograms and a numerical algorithm, which can retrieve phase maps of the samples with negligible errors under the ambient 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 newly developed algorithm with a high accuracy less than or equal to 5.5*10-4*pi 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. By using 12-bit CMOS camera the height of shallow rib waveguide is measured with a height sensitivity of 4 Angstrom without using PZT and in presence of environmental fluctuations.

physics.optics↗

arXiv2018

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. However, color CCD cameras are suffered from the color crosstalk issue, which needed to be explored. 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 of two different cameras commonly used in QPI, single chip CCD (1-CCD) and three chip CCD (3-CCD), is systematically studied and compared using compact interference microscopy. 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. It is demonstrated that the color crosstalk of the detector is the key limiting factor for phase measurement accuracy of simultaneous multi-spectral QPI systems.

physics.ins-det↗

arXiv2018

Study of longitudinal coherence properties of pseudo thermal light source as a function of source size and temporal coherence

In conventional OCT, broadband light sources are generally utilized to obtain high axial resolution due to their low temporal coherence (TC) length. Purely monochromatic (i.e., high TC length) light sources like laser cannot be implemented to acquire high resolution optically sectioned images of the specimen. Contrary to this, pseudo thermal light source having high TC and low spatial coherence (SC) property can be employed to achieve high axial resolution comparable to broadband light source. In the present letter, a pseudo thermal light source is synthesized by passing a purely monochromatic laser beam through a rotating diffuser. The longitudinal coherence (LC) property of the pseudo thermal light source is studied as a function of source size and TC length. The LC length of the synthesized light source decreased as the source size increased. It is found that LC length of such light source becomes independent of the parent laser TC length for source size of greater than or equal to 3.3 mm and become almost constant at around 30 micron for both the lasers. Thus any monochromatic laser light source can be utilized to obtain high axial resolution in OCT system irrespective of its TC length. The maximum achievable axial resolution is found to be equal to 650 nm corresponding to 1.2 numerical aperture (NA) objective lens at 632 nm wavelength. The findings elucidate that pseudo thermal source being monochromatic in nature can improve the performance of existing OCT systems significantly.

physics.optics↗