Common-path digital holographic microscopy based on a volume holographic grating for quantitative phase imaging.

Digital holographic microscopy (DHM) is a powerful quantitative phase imaging (QPI) technique that is capable of recording sample's phase information to enhance image contrast. In off-axis DHM, high-quality QPI images can be generated within a single recorded hologram, and the system stability can be enhanced by common-path configuration. Diffraction gratings are widely used components in common-path DHM systems; however, the presence of multiple diffraction beams leads to system power loss. Here, we propose and demonstrate implementation of a volume holographic grating (VHG) in common-path DHM, which provides single diffraction order. VHG in common-path DHM (i.e., VHG-DHM) helps in improving signal-to-noise ratio as compared to the conventional DHM. In addition, VHG, with inherently high angular selectivity, reduces image noise caused by stray light. With a simple fabrication process, it is convenient to utilize VHG to control the beam separation angle of DHM. Further, by using Bragg-matched wavelength degeneracy to avoid potential cell damaging effect in blue light, the VHG is designed for recording at a maximum sensitive wavelength of ∼488 nm, while our VHG-DHM is operated at the longer wavelength of red 632.8 nm for cell observation. Experimental results, measured by the VHG-DHM, show the measurement of target thickness ranging from 100 nm to 350 nm. In addition, stability of the system is quantitatively measured. High-contrast QPI images of human lung cancer cells are demonstrated.

Volume holographic illuminator for Airy light-sheet microscopy.

Airy light sheets combined with the deconvolution approach can provide multiple benefits, including large field of view (FOV), thin optical sectioning, and high axial resolution. The efficient design of an Airy light-sheet fluorescence microscope requires a compact illumination system. Here, we show that an Airy light sheet can be conveniently implemented in microscopy using a volume holographic grating (VHG). To verify the FOV and the axial resolution of the proposed VHG-based Airy light-sheet fluorescence microscope, ex-vivo fluorescently labeled Caenorhabditis elegans (C. elegans) embryos were imaged, and the Richardson-Lucy deconvolution method was used to improve the image contrast. Optimized parameters for deconvolution were compared with different methods. The experimental results show that the FOV and the axial resolution were 196 µm and 3 µm, respectively. The proposed method of using a compact VHG to replace the common spatial light modulator provides a direct solution to construct a compact light-sheet fluorescence microscope.

Abrupt autofocusing beam from a phase-only mask.

Airy beams have become an important beam shape for structured light beams because of their interesting self-accelerating and parabolic propagation properties. Many variants of Airy beams have been proposed, among which the Airy beam with cylindrical symmetry [also known as the circular Airy beam or abrupt autofocusing (AAF) beam] is particularly peculiar and has attracted special attention due to its shape transformation during propagation. Much effort has been devoted to understanding the properties of the AAF beam. In this work, we present simulation results for generating the AAF beam using a phase-only mask. A cubic chirp-modulated axicon phase is used to create the mask. We found an optimal value for the axiconic phase, and the cubic phase is essential for controlling the AAF beam's shape. We demonstrate that a phase-only mask is an effective and simple method for generating high contrast between the initial and AAF plane. We present the results for beam formation and propagation dynamics of the AAF beam using the control parameters of the phase mask. We also discuss the design parameters and their influence on the AAF beam shapes. Our results pave the way for a deeper understanding of the beam formation and propagation dynamics of the AAF beam.

Intelligent Phase Contrast Meta-Microscope System.

Phase contrast imaging techniques enable the visualization of disparities in the refractive index among various materials. However, these techniques usually come with a cost: the need for bulky, inflexible, and complicated configurations. Here, we propose and experimentally demonstrate an ultracompact meta-microscope, a novel imaging platform designed to accomplish both optical and digital phase contrast imaging. The optical phase contrast imaging system is composed of a pair of metalenses and an intermediate spiral phase metasurface located at the Fourier plane. The performance of the system in generating edge-enhanced images is validated by imaging a variety of human cells, including lung cell lines BEAS-2B, CLY1, and H1299 and other types. Additionally, we integrate the ResNet deep learning model into the meta-microscope to transform bright-field images into edge-enhanced images with high contrast accuracy. This technology promises to aid in the development of innovative miniature optical systems for biomedical and clinical applications.

Clinical outcomes of bronchiectasis in India: data from the EMBARC/Respiratory Research Network of India registry.

BACKGROUND: Identifying risk factors for poor outcomes can help with risk stratification and targeting of treatment. Risk factors for mortality and exacerbations have been identified in bronchiectasis but have been almost exclusively studied in European and North American populations. This study investigated the risk factors for poor outcome in a large population of bronchiectasis patients enrolled in India. METHODS: The European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India (EMBARC-India) registry is a prospective observational study of adults with computed tomography-confirmed bronchiectasis enrolled at 31 sites across India. Baseline characteristics of patients were used to investigate associations with key clinical outcomes: mortality, severe exacerbations requiring hospital admission, overall exacerbation frequency and decline in forced expiratory volume in 1 s. RESULTS: 1018 patients with at least 12-month follow-up data were enrolled in the follow-up study. Frequent exacerbations (≥3 per year) at baseline were associated with an increased risk of mortality (hazard ratio (HR) 3.23, 95% CI 1.39-7.50), severe exacerbations (HR 2.71, 95% CI 1.92-3.83), future exacerbations (incidence rate ratio (IRR) 3.08, 95% CI 2.36-4.01) and lung function decline. Coexisting COPD, dyspnoea and current cigarette smoking were similarly associated with a worse outcome across all end-points studied. Additional predictors of mortality and severe exacerbations were increasing age and cardiovascular comorbidity. Infection with Gram-negative pathogens (predominantly Klebsiella pneumoniae) was independently associated with increased mortality (HR 3.13, 95% CI 1.62-6.06), while Pseudomonas aeruginosa infection was associated with severe exacerbations (HR 1.41, 95% CI 1.01-1.97) and overall exacerbation rate (IRR 1.47, 95% CI 1.13-1.91). CONCLUSIONS: This study identifies risk factors for morbidity and mortality among bronchiectasis patients in India. Identification of these risk factors may support treatment approaches optimised to an Asian setting.

Self-supervised neural network for phase retrieval in QDPC microscopy.

Quantitative differential phase contrast (QDPC) microscope plays an important role in biomedical research since it can provide high-resolution images and quantitative phase information for thin transparent objects without staining. With weak phase assumption, the retrieval of phase information in QDPC can be treated as a linearly inverse problem which can be solved by Tikhonov regularization. However, the weak phase assumption is limited to thin objects, and tuning the regularization parameter manually is inconvenient. A self-supervised learning method based on deep image prior (DIP) is proposed to retrieve phase information from intensity measurements. The DIP model that takes intensity measurements as input is trained to output phase image. To achieve this goal, a physical layer that synthesizes the intensity measurements from the predicted phase is used. By minimizing the difference between the measured and predicted intensities, the trained DIP model is expected to reconstruct the phase image from its intensity measurements. To evaluate the performance of the proposed method, we conducted two phantom studies and reconstructed the micro-lens array and standard phase targets with different phase values. In the experimental results, the deviation of the reconstructed phase values obtained from the proposed method was less than 10% of the theoretical values. Our results show the feasibility of the proposed methods to predict quantitative phase with high accuracy, and no use of ground truth phase.

Volume holography-based abrupt autofocusing beam.

Volume holographic elements are excellent at shaping high-quality spatial and spectral modes. Many microscopy and laser-tissue interaction applications require precise delivery of optical energy at specific sites without affecting the peripheral regions. Owing to the property of very high energy contrast between the input and the focal plane, abrupt autofocusing (AAF) beams can be the right candidate for laser-tissue interaction. In this work, we demonstrate the recording and reconstruction of a PQ:PMMA photopolymer-based volume holographic optical beam shaper for an AAF beam. We experimentally characterize the generated AAF beams and show the broadband operation property. The fabricated volume holographic beam shaper shows long-term optical quality and stability. Our method offers multiple advantages including high angular selectivity, broadband operation, and intrinsically compact size. The present method may find important applications in designing compact optical beam shapers for biomedical lasers, illumination for microscopy, optical tweezers, and laser-tissue interaction experiments.

Multifocal confocal microscopy using a volume holographic lenslet array illuminator.

Multifocal illumination can improve image acquisition time compared to single point scanning in confocal microscopy. However, due to an increase in the system complexity, obtaining uniform multifocal illumination throughout the field of view with conventional methods is challenging. Here, we propose a volume holographic lenslet array illuminator (VHLAI) for multifocal confocal microscopy. To obtain uniform array illumination, a super Gaussian (SG) beam has been incorporated through VHLAI with an efficiency of 43%, and implemented in a confocal microscope. The design method for a photo-polymer based volume holographic beam shaper is presented and its advantages are thoroughly addressed. The proposed system can significantly improve image acquisition time without sacrificing the quality of the image. The performance of the proposed multifocal confocal microscopy was compared with wide-field images and also evaluated by measuring optically sectioned microscopic images of fluorescence beads, florescence pollen grains, and biological samples. The proposed multifocal confocal system generates images faster without any changes in scanning devices. The present method may find important applications in high-speed multifocal microscopy platforms.

Varifocal Metalens for Optical Sectioning Fluorescence Microscopy.

Fluorescence microscopy with optical sectioning capabilities is extensively utilized in biological research to obtain three-dimensional structural images of volumetric samples. Tunable lenses have been applied in microscopy for axial scanning to acquire multiplane images. However, images acquired by conventional tunable lenses suffer from spherical aberration and distortions. Here, we design, fabricate, and implement a dielectric Moiré metalens for fluorescence imaging. The Moiré metalens consists of two complementary phase metasurfaces, with variable focal length, ranging from ∼10 to ∼125 mm at 532 nm by tuning mutual angles. In addition, a telecentric configuration using the Moiré metalens is designed for high-contrast multiplane fluorescence imaging. The performance of our system is evaluated by optically sectioned images obtained from HiLo illumination of fluorescently labeled beads, as well as ex vivo mice intestine tissue samples. The compact design of the varifocal metalens may find important applications in fluorescence microscopy and endoscopy for clinical purposes.

Multi-plane confocal microscopy with multiplexed volume holographic gratings [Invited].

A volume holographic (VHG) grating-based multi-plane differential confocal microscopy (DCM) is proposed for axial scan-free imaging. Also, we briefly reviewed our previous works on volume holographic-based confocal imaging. We show that without degrading imaging performance, it is possible to simultaneously obtain two depth-resolved optically sectioned images with improved axial resolution using multi-plane DCM. The performance of our multi-plane DCM was evaluated by measuring the surface profile of a silicon micro-hole array with depths separation around 10 µm. The axial sensitivity of the system is around 25 nm. Our system has the advantages of multi-plane imaging with high axial sensitivity and high optical sectioning ability. Our method can be used for reflective surface profiling and multi-plane fluorescence imaging. The present methods may find important applications in surface metrology for label-free biological samples, as well as industrial applications.

Volume holographic optical element for light sheet fluorescence microscopy.

Three-dimensional (3D) imaging of living organisms requires fine optical sectioning and high-speed image acquisition, which can be achieved by light sheet fluorescence microscopy (LSFM). However, orthogonal illumination and detection arms in the LSFM system make it bulky. Here, we propose and demonstrate the application of a volume holographic optical element (photopolymer-based volume holographic grating) for designing a compact LSFM system, called a volume holographic LSFM (VHLSFM). Using the VHLSFM, we performed in vivo imaging of Caenorhabditis elegans (C. elegans) and observed high-contrast optically sectioned fluorescence images of the oocytes and embryonic development in real time for 3D imaging.

Multi-wavelength quantitative differential phase contrast imaging by radially asymmetric illumination.

A new approach for achieving isotropic differential phase contrast imaging by applying multi-wavelength asymmetric illumination is demonstrated. Multi-wavelength isotropic differential phase contrast scheme (MW-iDPC) can be implemented using an add-on module in any commercial inverted microscope. Isotropy of intensity transfer function is achieved using three axis measurements. The expression for MW-iDPC imaging is presented, and detailed mathematical analysis is performed for transfer function. By applying color leakage correction, image sensor responses can be calibrated. Asymmetric illumination masks are designed, and simulation studies for intensity of the transfer function are performed. We utilize the MW-iDPC system to reconstruct quantitative phase images of standard microspheres and live breast cancer cells. The optical thickness of cells can be measured with high accuracy and image acquisition time is reduced significantly.

Volume holographic spatial-spectral imaging systems [Invited].

In this paper, we present an overview of the recent developments in applications of volume holographic imaging techniques in microscopy. In these techniques, three-dimensional imaging incorporates multiplexed volume holographic gratings, which are formed in phenanthrenequinone poly(methyl methacrylate) (PQ-PMMA) photopolymer and act as spatial-spectral filters, to obtain multiplane images from a volumetric object without scanning. We introduce recent major roles of volume holography in different imaging modalities, including large-capacity spatial-spectral multiplane microscopy, digital holographic microscopy, and structured Talbot (or speckle) illumination fluorescence imaging. Among various imaging applications of volume holography, simultaneous multiplane fluorescence microscopy for collecting spatial-spectral information is distinct and has great potential for hyperspectral imaging. Depth selective spatial-spectral information from an object is particularly useful for designing a high-resolution microscope in real-time operation. We further discuss volume holography in particle trapping and beam shaping. In addition, we investigate future prospects of volume holography in microscopy as well as endoscopy.

Conventional volume holography for unconventional Airy beam shapes.

Utilizing multiplexed volume holography, a single optical element, enabling to shape structural variants of non-diffracting Airy wavefronts, from one-dimensional Airy mode to unconventional Airy modes such as vortex Airy and quad Airy modes, has been experimentally realized. Here, beam shaped angularly multiplexed volume holographic gratings (AMVHGs) are recorded in PQ: PMMA photopolymer, where five different spatial wavefronts of Airy beams have been sequentially recorded, for simultaneous reconstruction of different Airy modes, by a conventional Gaussian beam. Spatial and spectral mode selective properties of AMVHGs are demonstrated by narrow-band as well as by broadband light source. In addition, through wavelength degeneracy property, the maximum sensitivity wavelength of blue (488 nm) is used for recording in PQ: PMMA, but the AMVHGs are operated at a broad wavelength band of interest, all the way to longer wavelength in near infrared (850 nm). The K-sphere representation is used to explain the spectral properties of AMVHGs.

Spatial mode multiplexing using volume holographic gratings.

We experimentally demonstrate spatial mode multiplexing of optical beams using multiplexed volume holographic gratings (MVGHs) formed in phenanthrenquinone-poly (methyl methacrylate) (PQ-PMMA) photopolymer. Multiple spatial modes of Laguerre-Gaussian (LG) beams are recorded at the same pupil area of a volume hologram resulting in MVHGs, for simultaneous reconstruction of spatial modes. In addition, a helical phase beam, a non-diffracting beam with conical phase profile, and a parabolic non-diffracting beam with cubic phase profile have also been simultaneously recorded and reconstructed from MVHGs. Utilizing Bragg wavelength degeneracy property of volume hologram these multiplexed modes are reconstructed at multiple wavelengths ranging from blue (450nm) to red (635). Due to combined effect of three-dimensional pupil, Bragg wavelength degeneracy, angular selectivity, together with spatial mode properties these, MVHGs can act as spatial mode filter with spectral filtering property. Advantages of volume holography in beam shaping are discussed. Multiple first diffraction orders with desired beam shapes obtained from the single optical element (i.e. a volume hologram with MVHGs) may find important applications in optical communication experiments, and in volume holographic imaging and microscopy. Experimental results show solid evidence that MVGHs in beam shaping provide a simple, compact, single element, and direct way to multiplex spatial modes.

Creation of polarization gradients from superposition of counter propagating vector LG beams.

We present a detailed theoretical analysis of the formation of standing waves using cylindrically polarized vector Laguerre-Gaussian (LG) beams. It is shown that complex interplay between the radial and azimuthal polarization state can be used to realize different kinds of polarization gradients with cylindrically symmetric polarization distribution. Expressions for four different cases are presented and local dynamics of spatial polarization distribution is studied. We show cylindrically symmetric Sisyphus and corkscrew type polarization gradients can be obtained from vector LG beams. The optical landscape presented here with spatially periodic polarization patterns may find important applications in the field of atom optics, atom interferometry, atom lithography, and optical trapping.

Generation of radially polarized Bessel-Gaussian beams from c-cut Nd:YVO4 laser.

We experimentally demonstrate the generation of radially polarized Bessel-Gaussian beams from a c-cut Nd:YVO4 laser with a hemispherical cavity configuration by proper mode control. The output beam has an annular-shaped intensity distribution with radial polarization. When the beam is focused, the intensity pattern changes to a multi-ring, which is a typical characteristic of the lowest transverse mode of vector Bessel-Gaussian beam. Higher-order modes of vector Bessel-Gaussian beam are also observed from the same cavity by slightly changing the cavity alignment. The experimental results show a good agreement with the simulation results for both focal and far fields. The present method is a simple and direct way for generating vector Bessel-Gaussian beams.

Generation of a vector doughnut beam from an internal mirror He-Ne laser.

We demonstrate the direct generation of a vector doughnut mode from an internal mirror He-Ne laser using the spot defect method. A circular-shaped spot defect of ∼30 µm in diameter with low reflectivity created on the inner surface of a cavity mirror by laser ablation suppressed the oscillation of a Gaussian beam resulting in the oscillation of a doughnut beam. Polarization investigation showed that the generated beam was a vector hybrid mode.

Multiplane differential saturated excitation microscopy using varifocal lenses.

Saturated excitation microscopy, which collects nonlinear fluorescence signals generated by saturation, has been proposed to improve three-dimensional spatial resolution. Differential saturated excitation (dSAX) microscopy can further improve the detection efficiency of a nonlinear fluorescence signal. By comparing signals obtained at different saturation levels, high spatial resolution can be achieved in a simple and efficient manner. High-resolution multiplane microscopy is perquisite for volumetric imaging of thick samples. To the best of our knowledge, no reports of multiplane dSAX have been made. Our aim is to obtain multiplane high-resolution optically sectioned images by adapting differential saturated excitation in confocal laser scanning fluorescence microscopy. To perform multiplane dSAX microscopy, a variable focus lens is employed in a telecentric design to achieve focus tunability with constant magnification and contrast throughout the axial scanning range. Multiplane fluorescence imaging of two different types of pollen grains shows improved resolution and contrast. Our system's imaging performance is evaluated using standard targets, and the results are compared with standard confocal microscopy. Using a simple and efficient method, we demonstrate multiplane high-resolution fluorescence imaging. We anticipate that high-spatial resolution combined with high-speed focus tunability with invariant contrast and magnification will be useful in performing 3D imaging of thick biological samples.

In Vivo Intelligent Fluorescence Endo-Microscopy by Varifocal Meta-Device and Deep Learning.

Endo-microscopy is crucial for real-time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta-device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo-microscope is developed based on varifocal meta-lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta-lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field-of-view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high-resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc.