Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

[Simulation and Navigation in the Neurosurgical Field Using Three-Dimensional Hholograms by Mixed Reality Devices].

Recently, three-dimensional(3D)holograms from mixed-reality(MR)devices have become available in the medical field. 3D holographic images can provide immersive and intuitive information that has been reported to be very useful for preoperative simulations. Compared with conventional 3D images on a two-dimensional(2D)monitor, 3D holograms offer a higher level of realism, allowing observation of the images anytime and anywhere if the MR device is operational. Even during surgery, surgeons can check realistic 3D holograms in front of them, above the surgical field, without having to turn their heads toward a 2D monitor on the wall. 3D holograms can also be used for neuronavigation if the hologram is tracked to the patient's real head. This method can be defined as 3D augmented reality(AR)navigation, which shows a hologram of a target, such as a tumor or aneurysm, inside the head and brain. In the future, interventions using these techniques with 3D holograms from MR devices are expected to evolve and develop new types of treatments for endoscopic surgery or fluoroscopy-guided endovascular surgery.

3D polarization-interference holographic histology for wavelet-based differentiation of the polycrystalline component of biological tissues with different necrotic states. Forensic applications.

Significance: The interference-holographic method of phase scanning of fields of scattered laser radiation is proposed. The effectiveness of this method for the selection of variously dispersed components is demonstrated. This method made it possible to obtain polarization maps of biological tissues at a high level of depolarized background. The scale-selective analysis of such maps was used to determine necrotic changes in the optically anisotropic architectonics of biological tissues. Objective: Development and experimental approbation of layered phase polarimetry of repeatedly scattered fields in diffuse layers of biological tissues. Application of scale-selective processing of the found coordinate distributions of polarization states in various phase sections of object fields. Determination of criteria (markers) for histological differential diagnosis of the causes of necrotic changes in optical anisotropy of biological tissues. Approach: We used a synthesis of three instrumental and analytical methods. Polarization-interference registration of laser radiation scattered by a sample of biological tissue. Digital holographic reconstruction and layered phase scanning of distributions of complex amplitudes of the object field. Analytical determination of polarization maps of various phase cross-sections of repeatedly scattered radiation. Application of wavelet analysis of the distributions of polarization states in the phase plane of a single scattered component of an object field. Determination of criteria (markers) for differential diagnosis of necrotic changes in biological tissues with different morphological structure. Two cases are considered. The first case is the myocardium of those who died as a result of coronary heart disease and acute coronary insufficiency. The second case is lung tissue samples of deceased with bronchial asthma and fibrosis. Results: A method of polarization-interference mapping of diffuse object fields of biological tissues has been developed and experimentally implemented. With the help of digital holographic reconstruction of the distributions of complex amplitudes, polarization maps in various phase sections of a diffuse object field are found. The wavelet analysis of azimuth and ellipticity distributions of polarization in the phase plane of a single scattered component of laser radiation is used. Scenarios for changing the amplitude of the wavelet coefficients for different scales of the scanning salt-like MHAT function are determined. Statistical moments of the first to fourth orders are determined for the distributions of the amplitudes of the wavelet coefficients of the azimuth maps and the ellipticity of polarization. As a result, diagnostic markers of necrotic changes in the myocardium and lung tissue were determined. The statistical criteria found are the basis for determining the accuracy of their differential diagnosis of various necrotic states of biological tissues. Conclusions: Necrotic changes caused by "coronary artery disease-acute coronary insufficiency" and "asthma-pulmonary fibrosis" were demonstrated by the method of wavelet differentiation with polarization interference with excellent accuracy.

Comparative oncology chemosensitivity assay for personalized medicine using low-coherence digital holography of dynamic light scattering from cancer biopsies.

Nearly half of cancer patients who receive standard-of-care treatments fail to respond to their first-line chemotherapy, demonstrating the pressing need for improved methods to select personalized cancer therapies. Low-coherence digital holography has the potential to fill this need by performing dynamic contrast OCT on living cancer biopsies treated ex vivo with anti-cancer therapeutics. Fluctuation spectroscopy of dynamic light scattering under conditions of holographic phase stability captures ultra-low Doppler frequency shifts down to 10 mHz caused by light scattering from intracellular motions. In the comparative preclinical/clinical trials presented here, a two-species (human and canine) and two-cancer (esophageal carcinoma and B-cell lymphoma) analysis of spectral phenotypes identifies a set of drug response characteristics that span species and cancer type. Spatial heterogeneity across a centimeter-scale patient biopsy sample is assessed by measuring multiple millimeter-scale sub-samples. Improved predictive performance is achieved for chemoresistance profiling by identifying red-shifted sub-samples that may indicate impaired metabolism and removing them from the prediction analysis. These results show potential for using biodynamic imaging for personalized selection of cancer therapy.

Live Cell Imaging by Single-Shot Common-Path Wide Field-of-View Reflective Digital Holographic Microscope.

Quantitative phase imaging by digital holographic microscopy (DHM) is a nondestructive and label-free technique that has been playing an indispensable role in the fields of science, technology, and biomedical imaging. The technique is competent in imaging and analyzing label-free living cells and investigating reflective surfaces. Herein, we introduce a new configuration of a wide field-of-view single-shot common-path off-axis reflective DHM for the quantitative phase imaging of biological cells that leverages several advantages, including being less-vibration sensitive to external perturbations due to its common-path configuration, also being compact in size, simple in optical design, highly stable, and cost-effective. A detailed description of the proposed DHM system, including its optical design, working principle, and capability for phase imaging, is presented. The applications of the proposed system are demonstrated through quantitative phase imaging results obtained from the reflective surface (USAF resolution test target) as well as transparent samples (living plant cells). The proposed system could find its applications in the investigation of several biological specimens and the optical metrology of micro-surfaces.

Holographic Brain Theory: Super-Radiance, Memory Capacity and Control Theory.

We investigate Quantum Electrodynamics corresponding to the holographic brain theory introduced by Pribram to describe memory in the human brain. First, we derive a super-radiance solution in Quantum Electrodynamics with non-relativistic charged bosons (a model of molecular conformational states of water) for coherent light sources of holograms. Next, we estimate memory capacity of a brain neocortex, and adopt binary holograms to manipulate optical information. Finally, we introduce a control theory to manipulate holograms involving biological water's molecular conformational states. We show how a desired waveform in holography is achieved in a hierarchical model using numerical simulations.

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy.

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes - the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines - the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.

A dataset of digital holograms of normal and thalassemic cells.

Digital holographic microscopy (DHM) is an intriguing medical diagnostic tool due to its label-free and quantitative nature, providing high-contrast images of phase samples. By capturing both intensity and phase information, DHM enables the numerical reconstruction of quantitative phase images. However, the lateral resolution is limited by the diffraction limit, which prompted the recent suggestion of microsphere-assisted DHM to enhance the DHM resolution straightforwardly. The use of such a technique as a medical diagnostic tool requires testing and validation of the proposed assays to prove their feasibility and viability. This paper publishes 760 and 609 microsphere-assisted DHM images of normal and thalassemic red blood cells obtained from a normal and thalassemic male individual, respectively.

Label-free cell classification in holographic flow cytometry through an unbiased learning strategy.

Nowadays, label-free imaging flow cytometry at the single-cell level is considered the stepforward lab-on-a-chip technology to address challenges in clinical diagnostics, biology, life sciences and healthcare. In this framework, digital holography in microscopy promises to be a powerful imaging modality thanks to its multi-refocusing and label-free quantitative phase imaging capabilities, along with the encoding of the highest information content within the imaged samples. Moreover, the recent achievements of new data analysis tools for cell classification based on deep/machine learning, combined with holographic imaging, are urging these systems toward the effective implementation of point of care devices. However, the generalization capabilities of learning-based models may be limited from biases caused by data obtained from other holographic imaging settings and/or different processing approaches. In this paper, we propose a combination of a Mask R-CNN to detect the cells, a convolutional auto-encoder, used to the image feature extraction and operating on unlabelled data, thus overcoming the bias due to data coming from different experimental settings, and a feedforward neural network for single cell classification, that operates on the above extracted features. We demonstrate the proposed approach in the challenging classification task related to the identification of drug-resistant endometrial cancer cells.

Augmented Reality Holographic Visualization System for Surgery Auxiliary Visualization: Proof of Concept for Surgical Training.

An Augmented Reality (AR) system based on the holographic projection of the relevant anatomic structures is proposed for auxiliary visualization during surgeries. The current two-dimensional visualization systems require the surgeons to mentally extract the associated three-dimensional information during the interventions, which entails risks and complications. This work shows an AR holographic projection system for real-time three-dimensional representation of the relevant surgical information, thus overcoming this problem. As an initial proof of concept, the system is experimentally assessed as potential surgery training tool.Clinical Relevance- This work explores the potential of AR holographic projection systems for intraoperative assistance to the surgical team, starting from its possible use as surgery training and planning tool.

Fast 2-photon stimulation using holographic patterns.

Two decades after its introduction, optogenetics - a biological technique to control the activity of neurons or other cell types with light - remains a cutting edge and promising tool to study biological processes. Its increasing usage in research varies widely from causally exploring biological mechanisms and neural computations, to neurostimulation and sensory restauration. To stimulate neurons in the brain, a variety of approaches have been developed to generate precise spatiotemporal light patterns. Yet certain constrains still exists in the current optical techniques to activate a neuronal population with both cellular resolution and millisecond precision. Here, we describe an experimental setup allowing to stimulate a few tens of neurons in a plane at sub-millisecond rates using 2-photon activation. A liquid crystal on silicon spatial light modulator (LCoS-SLM) was used to generate spatial patterns in 2 dimensions. The image of the patterns was formed on the plane of a digital micromirror device (DMD) that was used as a fast temporal modulator of each region of interest. Using fluorescent microscopy and patch-clamp recording of neurons in culture expressing the light-gated ion channels, we characterized the temporal and spatial resolution of the microscope. We described the advantages of combining the LCoS-SLM with the DMD to maximize the temporal precision, modulate the illumination amplitude, and reduce background activation. Finally, we showed that this approach can be extended to patterns in 3 dimensions. We concluded that the methodology is well suited to address important questions about the role of temporal information in neuronal coding.

Aberration-free holographic microscope for simultaneous imaging and stimulation of neuronal populations.

A technical challenge in neuroscience is to record and specifically manipulate the activity of neurons in living animals. This can be achieved in some preparations with two-photon calcium imaging and photostimulation. These methods can be extended to three dimensions by holographic light sculpting with spatial light modulators (SLMs). At the same time, performing simultaneous holographic imaging and photostimulation is still cumbersome, requiring two light paths with separate SLMs. Here we present an integrated optical design using a single SLM for simultaneous imaging and photostimulation. Furthermore, we applied axially dependent adaptive optics to make the system aberration-free, and developed software for calibrations and closed-loop neuroscience experiments. Finally, we demonstrate the performance of the system with simultaneous calcium imaging and optogenetics in mouse primary auditory cortex in vivo. Our integrated holographic system could facilitate the systematic investigation of neural circuit function in awake behaving animals.

Fluorescence-guided surgical system using holographic display: from phantom studies to canine patients.

Significance: Holographic display technology is a promising area of research that can lead to significant advancements in cancer surgery. We present the benefits of combining bioinspired multispectral imaging technology with holographic goggles for fluorescence-guided cancer surgery. Through a series of experiments with 43D-printed phantoms, small animal models of cancer, and surgeries on canine patients with head and neck cancer, we showcase the advantages of this holistic approach. Aim: The aim of our study is to demonstrate the feasibility and potential benefits of utilizing holographic display for fluorescence-guided surgery through a series of experiments involving 3D-printed phantoms and canine patients with head and neck cancer. Approach: We explore the integration of a bioinspired camera with a mixed reality headset to project fluorescent images as holograms onto a see-through display, and we demonstrate the potential benefits of this technology through benchtop and in vivo animal studies. Results: Our complete imaging and holographic display system showcased improved delineation of fluorescent targets in phantoms compared with the 2D monitor display approach and easy integration into the veterinarian surgical workflow. Conclusions: Based on our findings, it is evident that our comprehensive approach, which combines a bioinspired multispectral imaging sensor with holographic goggles, holds promise in enhancing the presentation of fluorescent information to surgeons during intraoperative scenarios while minimizing disruptions.

Multispectral in-line hologram reconstruction with aberration compensation applied to Gram-stained bacteria microscopy.

In multispectral digital in-line holographic microscopy (DIHM), aberrations of the optical system affect the repeatability of the reconstruction of transmittance, phase and morphology of the objects of interest. Here we address this issue first by model fitting calibration using transparent beads inserted in the sample. This step estimates the aberrations of the optical system as a function of the lateral position in the field of view and at each wavelength. Second, we use a regularized inverse problem approach (IPA) to reconstruct the transmittance and phase of objects of interest. Our method accounts for shift-variant chromatic and geometrical aberrations in the forward model. The multi-wavelength holograms are jointly reconstructed by favouring the colocalization of the object edges. The method is applied to the case of bacteria imaging in Gram-stained blood smears. It shows our methodology evaluates aberrations with good repeatability. This improves the repeatability of the reconstructions and delivers more contrasted spectral signatures in transmittance and phase, which could benefit applications of microscopy, such as the analysis and classification of stained bacteria.

Comprehensive tool for a phase compensation reconstruction method in digital holographic microscopy operating in non-telecentric regime.

Quantitative phase imaging (QPI) via Digital Holographic microscopy (DHM) has been widely applied in material and biological applications. The performance of DHM technologies relies heavily on computational reconstruction methods to provide accurate phase measurements. Among the optical configuration of the imaging system in DHM, imaging systems operating in a non-telecentric regime are the most common ones. Nonetheless, the spherical wavefront introduced by the non-telecentric DHM system must be compensated to provide undistorted phase measurements. The proposed reconstruction approach is based on previous work from Kemper's group. Here, we have reformulated the problem, reducing the number of required parameters needed for reconstructing phase images to the sensor pixel size and source wavelength. The developed computational algorithm can be divided into six main steps. In the first step, the selection of the +1-diffraction order in the hologram spectrum. The interference angle is obtained from the selected +1 order. Secondly, the curvature of the spherical wavefront distorting the sample's phase map is estimated by analyzing the size of the selected +1 order in the hologram's spectrum. The third and fourth steps are the spatial filtering of the +1 order and the compensation of the interference angle. The next step involves the estimation of the center of the spherical wavefront. An optional final optimization step has been included to fine-tune the estimated parameters and provide fully compensated phase images. Because the proper implementation of a framework is critical to achieve successful results, we have explicitly described the steps, including functions and toolboxes, required for reconstructing phase images without distortions. As a result, we have provided open-access codes and a user interface tool with minimum user input to reconstruct holograms recorded in a non-telecentric DHM system.

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.

Structured illumination phase and fluorescence microscopy for bioimaging.

This study presents a dual-modality microscopic imaging approach that combines quantitative phase microscopy and fluorescence microscopy based on structured illumination (SI) to provide structural and functional information for the same sample. As the first imaging modality, structured illumination digital holographic microscopy (SI-DHM) is implemented along the transmission beam path. SI-DHM acts as a label-free, noninvasive approach and provides high-contrast and quantitative phase images utilizing the refractive index contrast of the inner structures of samples against the background. As the second imaging modality, structured illumination (fluorescence) microscopy (SIM) is constructed along the reflection beam path. SIM utilizes fluorescent labeling and provides super-resolution images for specific functional structures of samples. We first experimentally demonstrated phase imaging of SI-DHM on rice leaves and fluorescence (SIM) imaging on mouse kidney sections. Then, we demonstrated dual-modality imaging of biological samples, using DHM to acquire the overall cell morphology and SIM to obtain specific functional structures. These results prove that the proposed technique is of great importance in biomedical studies, such as providing insight into cell physiology by visualizing and quantifying subcellular structures.

State-of-the-Art Electron Microscopy For Physical Sciences Research.

ARTICLES DISCUSSED: Moon, T., Colletta, M., Kourkoutis, L. F. Nanoscale characterization of liquid-solid interfaces by couple cryo-focused ion beam milling with scanning electron microscopy and spectroscopy. Journal of Visualized Experiments. (185), e61955 (2022). Ohtsuka, M., Muto, S. Quantitative atomic-site analysis of functional dopants/point defects in crystalline materials by electron-channeling-enhanced microanalysis. Journal of Visualized Experiments. (171), e62015 (2021). Miao, L., Chmielewski, A., Mukherjee, D., Alem, N. Picometer-precision atomic position tracking through electron microscopy. Journal of Visualized Experiments. (173), e62164 (2021). Unocic, K. A. et al. Performing in situ closed-cell gas reactions in the transmission electron microscope. Journal of Visualized Experiments. (173), e62174 (2021). Zheng, F. et al. Magnetic field mapping using off-axis electron holography in the transmission electron microscope. Journal of Visualized Experiments. (166), e61907 (2020).

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Integrated lithium niobate optical phased array for two-dimensional beam steering.

Optical phased arrays (OPAs) with high speed, low power consumption, and low insertion loss are appealing for various applications, including light detection and ranging, free-space communication, image projection, and imaging. These OPAs can be achieved by fully harnessing the advantages of integrated lithium niobate (LN) photonics, which include high electro-optical modulation speed, low driving voltage, and low optical loss. Here we present an integrated LN OPA that operates in the near-infrared regime. Our experimental results demonstrate 24 × 8° two-dimensional beam steering, a far-field beam spot with a full width at half maximum of 2 × 0.6°, and a sidelobe suppression level of 10 dB. Furthermore, the phase modulator of our OPA exhibits a half-wave voltage of 6 V. The low power consumption exhibited by our OPA makes it highly attractive for a wide range of applications. Beyond conventional applications, our OPA's high speed opens up the possibility of novel applications such as high-density point cloud generation and tomographic holography.