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.

Single-shot multispectral quantitative phase imaging of biological samples using deep learning.

Multispectral quantitative phase imaging (MS-QPI) is a high-contrast label-free technique for morphological imaging of the specimens. The aim of the present study is to extract spectral dependent quantitative information in single-shot using a highly spatially sensitive digital holographic microscope assisted by a deep neural network. There are three different wavelengths used in our method: λ=532, 633, and 808 nm. The first step is to get the interferometric data for each wavelength. The acquired datasets are used to train a generative adversarial network to generate multispectral (MS) quantitative phase maps from a single input interferogram. The network was trained and validated on two different samples: the optical waveguide and MG63 osteosarcoma cells. Validation of the present approach is performed by comparing the predicted MS phase maps with numerically reconstructed (F T+T I E) phase maps and quantifying with different image quality assessment metrices.

Homogeneous distribution of phosphor particles inside resin using a vertical vibration method for efficient laser-based white light illumination.

We report what we believe to be an innovative method for the homogeneous distribution of phosphor particles inside the optical resin-based layer using a vertical vibrational technique for efficient laser-based white light illumination. In this method, single-stage vibration energy was efficiently used against phosphor sedimentation with the help of a mechanical vibrator system. The vertical vibrational energy was transferred to negate the downward gravitational effect acting on each phosphor particle. Therefore, the phosphor particles inside the layer were stable and uniformly distributed from the initial to final drying stages, creating approximate refractive index homogeneity inside the medium. The sedimentation problem was resolved, and all the optical parameters to support this method were properly analyzed and found to be stable and suitable for laser-based illumination applications.

Fabrication of low cost highly structured silver capped aluminium nanorods as SERS substrate for the detection of biological pathogens.

We report the fabrication of low cost highly structured silver (Ag) capped aluminium (Al) nanorods (NRs) as surface enhanced Raman spectroscopy (SERS) substrate utilising the glancing angle deposition technique. The nano-capping of silver onto the Al NRs can concentrate the local electric field within the minimal volume that can serve as hotspots. The average size of the Ag nanocaps was 50 nm. The newly proposed nanoporous Ag capped Al NRs as SERS substrate could detect the Raman signal of rhodamine 6G (R6G) up to 10-15molar concentration. The significant enhancement in the Raman signal of 107was achieved for Ag capped Al NRs considering R6G as a probe molecule. Using the developed SERS substrate, we recorded Raman spectra forEscherichia colibacteria with its concentration varying from 108colony forming units per ml (CFU ml-1) up to 102CFU ml-1. All the reported Raman spectra were acquired by a portable handheld Raman spectrometer. Hence, this newly proposed low cost, effective SERS substrate can be used commercially for the onsite detection of clinical pathogens. The 3D finite difference time domain simulation model was performed for Ag capped Al nanostructure to understand the generation of hotspots. The simulated results show excellent agreement with the experimental results. We fabricated uncapped Ag nanorods of similar dimensions and performed the experimental measurements and simulations for comparison. We found a significant enhancement in Ag capped Al NRs compared to the long Ag NRs. The description of the Raman signal enhancement has been elaborated.

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.

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.

Laser-line-driven phosphor-converted extended white light source with uniform illumination.

We report the development of laser-driven extended white light source designed as a light sheet for general illumination. This light sheet is made of two large diffused glass plates. A Ce:YAG phosphor layer was coated and sandwiched between the two diffusing sheets. The blue laser beam was first converted into a uniform laser line using a cylindrical lens, and a laser line was made incident parallel to the edges of the designed light sheet. The blue photons are waveguided inside the glass sheet via total internal reflection and scattered from the diffused surface. Some of the blue photons are absorbed by the Ce:YAG phosphor and down-converted into yellow light of longer wavelength. The white light emanating from the diffuse surface is the combined effect of yellow light with original blue light. The developed light sheet with the combination of laser line generation and total internal reflection is a unique and low-cost method for generating white light with uniform illumination. The details of the development of the light sheet and laser line generation are described. The experimental parameters, such as correlated color temperature and color coordinates, are reported.

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.

Conical light sword optical beam and its healing property.

A new diffractive optical element, named as a conical light sword optical element, is presented. In the focal volume, this element produces a helical amplitude profile that can be used as an optical twister. We have experimentally demonstrated the optical healing property of the conical light sword optical beam (CLSOB). This healing property comes from the transverse helical energy flow, due to the evolution of multiple unipolar vortices in the propagation of CLSOB. We envisage that this spiral intensity profile and optical healing property of the beam find potential applications in propagation through a scattering and turbulent media, imaging with extended depth of field, and in optical tweezers.

High-resolution cell imaging using white light phase shifting interferometry and iterative phase deconvolution.

An optimization algorithm is presented for the deconvolution of a complex field to improve the resolution and accuracy of quantitative phase imaging (QPI). A high-resolution phase map can be recovered by solving a constrained optimization problem of deconvolution using a complex gradient operator. The method is demonstrated on phase measurements of samples using a white light based phase shifting interferometry (WLPSI) method. The application of the algorithm on real and simulated objects shows a significant resolution and contrast improvement. Experiments performed on Escherichia coli bacterium have revealed its sub-cellular structures that were not visible in the raw WLPSI images obtained using a five phase shifting method. These features can give valuable insights into the structures and functioning of biological cells. The algorithm is simple in implementation and can be incorporated into other QPI modalities .

Birefringence mapping of biological tissues based on polarization sensitive non-interferometric quantitative phase imaging technique.

OBJECTIVE: Oral cancer is a leading cause of mortality globally, particularly affecting developing regions where oral hygiene is often overlooked. The optical properties of tissues are vital for diagnostics, with polarization imaging emerging as a label-free, contrast-enhancing technique widely employed in medical and scientific research over past few decades. MATERIALS AND METHODS: We present a novel polarization sensitive quantitative phase imaging of biological tissues by incorporating the conventional polarization microscope and transport of intensity equation-based phase retrieval algorithm. This integration provides access to the birefringence mapping of biological tissues. The inherent optical anisotropy in biological tissues induces the polarization dependent refractive index variations which can provide the detailed insights into the birefringence characteristics of their extracellular constituents. Experimental investigations were conducted on both normal and cancerous oral tissue samples by recording a set of three polarization intensity images for each case with a step size of 2 µm. RESULTS: A noteworthy increment in birefringence quantification was observed in cancerous as compared to the normal tissues, attributed to the proliferation of abnormal cells during cancer progression. The mean birefringence values were calculated for both normal and cancerous tissues, revealing a significant increase in birefringence of cancerous tissues (2.1 ± 0.2) × 10-2 compared to normal tissues (0.8 ± 0.2) × 10-2. Data were collected from 8 patients in each group under identical experimental conditions. CONCLUSION: This polarization sensitive non-interferometric optical approach demonstrated effective discrimination between cancerous and normal tissues, with various parameters indicating elevated values in cancerous tissues.

Multispectral polarization microscopy of different stages of human oral tissue: A polarization study.

Many optical techniques have been used in various diagnostics and biomedical applications since a decade and polarization imaging is one of the non-invasive and label free optical technique to investigate biological samples making it an important tool in diagnostics, biomedical applications. We report a multispectral polarization-based imaging of oral tissue by utilizing a polarization microscope system with a broadband-light source. Experiments were performed on oral tissue samples and multispectral Stokes mapping was done by recording a set of intensity images. Polarization-based parameters like degree of polarization, angle of fast axis, retardation and linear birefringence have been retrieved. The statistical moments of these polarization components have also been reported at multiples wavelengths. The polarimetric properties of oral tissue at different stages of cancer have been analyzed and significant changes from normal to pre-cancerous lesions to the cancerous are observed in linear birefringence quantification as (1.7 ± 0.1) × 10-3 , (2.5 ± 0.2) × 10-3 and (3.3 ± 0.2) × 10-3 respectively.