Coherence Microscopy Research Articles

Resolution Improvement for Coherent Illumination Microscopy via Incident Light Phase Modulation

Resolution Improvement for Coherent Illumination Microscopy via Incident Light Phase Modulation

Gaussian coherent transfer function of partially coherent Fourier ptychographic microscopy

Gaussian coherent transfer function of partially coherent Fourier ptychographic microscopy

Fourier Ptychographic Coherent Anti-Stokes Raman Scattering Microscopy with Point-Scanning for Super-Resolution Imaging.

Fourier ptychography (FP) is a high resolution wide-field imaging method based on the extended aperture in the Fourier space, which is synthesized from raw images with varying illumination angles. If FP is extended to coherent nonlinear optical imaging, the resolution could be further improved due to the increase of the cutoff frequency of the synthesized coherent optical transfer function (C-OTF) with respect to the order of nonlinear optical processes. However, there is a fundamental conflict between wide-field FP and nonlinear optical imaging, whereby the nonlinear optical imaging typically requires a focused excitation laser beam with high power density. To tackle the problem, in this work, a unique point-scanning FP (PS-FP) method is presented for super-resolution nonlinear optical imaging, in which the nonlinear optical signal is obtained by using focused laser beam, while the conventional FP algorithm can still be used to retrieve the super-resolution image. PS-FP coherent anti-Stokes Raman scattering (PS-FP-CARS) imaging on a variety of samples, where a 1.8-fold expansion of the OTF is achieved experimentally for enhancing vibrational imaging. Further theoretical calculation shows that the C-OTF of PS-FP higher-order CARS (PS-FP-HO-CARS) can be expanded up to ≈4.9-fold, thereby improving the spatial resolution by ≈3-fold in comparison with conventional point-scanning CARS with under tightly focused beams. The generality of PS-FP method developed in this work can be adapted to other coherent nonlinear optical imaging modalities for super-resolution imaging in tissue and cells.

Photothermal Optical Coherence Microscopy for studying lipid architecture in human carotid arteries

Photothermal Optical Coherence Microscopy for studying lipid architecture in human carotid arteries

Dual color optogenetic tool enables heart arrest, bradycardic, and tachycardic pacing in Drosophila melanogaster

In order to facilitate cardiovascular research to develop non-invasive optical heart pacing methods, we have generated a double-transgenic Drosophila melanogaster (fruit fly) model suitable for optogenetic pacing. We created a fly stock with both excitatory H134R-ChR2 and inhibitory eNpHR2.0 opsin transgenes. Opsins were expressed in the fly heart using the Hand-GAL4 driver. Here we describe Hand > H134R-ChR2; eNpHR2.0 model characterization including bi-directional heart control (activation and inhibition) upon illumination of light with distinct wavelengths. Optical control and real-time visualization of the heart function were achieved non-invasively using an integrated light stimulation and optical coherence microscopy (OCM) system. OCM produced high-speed and high-resolution imaging; simultaneously, the heart function was modulated by blue (470 nm) or red (617 nm) light pulses causing tachycardia, bradycardia and restorable cardiac arrest episodes in the same animal. The irradiance power levels and illumination schedules were optimized to achieve successful non-invasive bi-directional heart pacing in Drosophila larvae and pupae.

Full-field optical coherence microscopy enables high-resolution label-free imaging of the dynamics of live mouse oocytes and early embryos

High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction.

Tumor spheroid elasticity estimation using mechano-microscopy combined with a conditional generative adversarial network

Background and ObjectivesTechniques for imaging the mechanical properties of cells are needed to study how cell mechanics influence cell function and disease progression. Mechano-microscopy (a high-resolution variant of compression optical coherence elastography) generates elasticity images of a sample undergoing compression from the phase difference between optical coherence microscopy (OCM) B-scans. However, the existing mechano-microscopy signal processing chain (referred to as the algebraic method) assumes the sample stress is uniaxial and axially uniform, such that violation of these assumptions reduces the accuracy and precision of elasticity images. Furthermore, it does not account for prior information regarding the sample geometry or mechanical property distribution. In this study, we investigate the feasibility of training a conditional generative adversarial network (cGAN) to generate elasticity images from phase difference images of samples containing a cell spheroid embedded in a hydrogel. MethodsTo construct the cGAN training and simulated test sets, we generated 30,000 artificial elasticity images using a parametric model and computed the corresponding phase difference images using finite element analysis to simulate compression applied to the artificial samples. We also imaged real MCF7 breast tumor spheroids embedded in hydrogel using mechano-microscopy to construct the experimental test set and evaluated the cGAN using the algebraic elasticity images and co-registered OCM and confocal fluorescence microscopy (CFM) images. ResultsComparison with the simulated test set ground truth elasticity images shows the cGAN produces a lower root mean square error (median: 3.47 kPa, 95 % confidence interval (CI) [3.41, 3.52]) than the algebraic method (median: 4.91 kPa, 95 % CI [4.85, 4.97]). For the experimental test set, the cGAN elasticity images contain features resembling stiff nuclei at locations corresponding to nuclei seen in the algebraic elasticity, OCM, and CFM images. Furthermore, the cGAN elasticity images are higher resolution and more robust to noise than the algebraic elasticity images. ConclusionsThe cGAN elasticity images exhibit better accuracy, spatial resolution, sensitivity, and robustness to noise than the algebraic elasticity images for both simulated and real experimental data.

Dispersion mismatch correction for evident chromatic anomaly in low coherence interferometry.

The applications of ultrafast optics to biomedical microscopy have expanded rapidly in recent years, including interferometric techniques like optical coherence tomography and microscopy (OCT/OCM). The advances of ultra-high resolution OCT and the inclusion of OCT/OCM in multimodal systems combined with multiphoton microscopy have marked a transition from using pseudo-continuous broadband sources, such as superluminescent diodes, to ultrafast supercontinuum optical sources. We report anomalies in the dispersion profiles of low-coherence ultrafast pulses through long and non-identical arms of a Michelson interferometer that are well beyond group delay or third-order dispersions. This chromatic anomaly worsens the observed axial resolution and causes fringe artifacts in the reconstructed tomograms in OCT/OCM using traditional algorithms. We present DISpersion COmpensation Techniques for Evident Chromatic Anomalies (DISCOTECA) as a universal solution to address the problem of chromatic dispersion mismatch in interferometry, especially with ultrafast sources. First, we demonstrate the origin of these artifacts through the self-phase modulation of ultrafast pulses due to focusing elements in the beam path. Next, we present three solution paradigms for DISCOTECA: optical, optoelectronic, and computational, along with quantitative comparisons to traditional methods to highlight the improvements to the dynamic range and axial profile. We explain the piecewise reconstruction of the phase mismatch between the arms of the spectral-domain interferometer using a modified short-term Fourier transform algorithm inspired by spectroscopic OCT. Finally, we present a decision-making guide for evaluating the utility of DISCOTECA in interferometry and for the artifact-free reconstruction of OCT images using an ultrafast supercontinuum source for biomedical applications.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

Digital histological staining of tissue slide images from optical coherence microscopy.

The convergence of staining-free optical imaging and digital staining technologies has become a central focus in digital pathology, presenting significant advantages in streamlining specimen preparation and expediting the rapid acquisition of histopathological information. Despite the inherent merits of optical coherence microscopy (OCM) as a staining-free technique, its widespread application in observing histopathological slides has been constrained. This study introduces a novel approach by combining wide-field OCM with digital staining technology for the imaging of histopathological slides. Through the optimization of the histology slide production process satisfying the ground growth for digital staining as well as pronounced contrast for OCM imaging, successful imaging of various mouse tissues was achieved. Comparative analyses with conventional staining-based bright field images were executed to evaluate the proposed methodology's efficacy. Moreover, the study investigates the generalization of digital staining color appearance to ensure consistent histopathology, considering tissue-specific and thickness-dependent variations.

Attention LSTM U-Net model for Drosophila melanogaster heart tube segmentation in optical coherence microscopy images.

Optical coherence microscopy (OCM) imaging of the Drosophila melanogaster (fruit fly) heart tube has enabled the non-invasive characterization of fly heart physiology in vivo. OCM generates large volumes of data, making it necessary to automate image analysis. Deep-learning-based neural network models have been developed to improve the efficiency of fly heart image segmentation. However, image artifacts caused by sample motion or reflections reduce the accuracy of the analysis. To improve the precision and efficiency of image data analysis, we developed an Attention LSTM U-Net model (FlyNet3.0), which incorporates an attention learning mechanism to track the beating fly heart in OCM images. The new model has improved the intersection over union (IOU) compared to FlyNet2.0 + with reflection artifacts from 86% to 89% and with movement from 81% to 89%. We also extended the capabilities of OCM analysis through the introduction of an automated, in vivo heart wall thickness measurement method, which has been validated on a Drosophila model of cardiac hypertrophy. This work will enable the comprehensive, non-invasive characterization of fly heart physiology in a high-throughput manner.

Characteristics and utility of high-resolution optical coherence microscopy images of endocervical canal lesions.

To investigate optical coherence microscopy (OCM) imaging features and the application value of these high-resolution images for identifying endocervical canal lesions (ECLs), which is a clinical dilemma in cervical cancer screening programs. In total, 520 OCM images were obtained by scanning the cervical canal lesions with an ultra-high-resolution OCM system (204 specimens from 73 patients). The OCM morphologic characteristics of ECLs were observed and summarized, and then 3 researchers performed a diagnostic test of OCM images of cervical canal lesions. The accuracy, sensitivity, specificity, positive predictive value, negative predictive value, 95% confidence interval of each parameter, and interinvestigator agreement (κ) were calculated. Normal endocervix, cysts, squamous metaplasia, high-grade squamous intraepithelial lesions involving glands, and invasive carcinoma had distinct OCM characteristics, which correlated well with corresponding H&E histologic sections. The accuracy, sensitivity, and specificity of the 3 researchers were 90.6%, 89.3% (95% CI, 86.5%-91.7%) and 91.6% (95% CI, 89.2%-93.5%), respectively. The positive predictive value was 90.1% (95% CI, 87.3%-92.4%), and the negative predictive value was 90.9% (95% CI, 88.5%-92.9%), with almost perfect agreement (κ = 0.874). The application of the OCM system in cervical canal lesions is feasible and could help improve detection of occult ECLs in cervical cancer screening programs. This study lays the foundation for further research on OCM in cervical canal lesions in vivo, which also has a potential impact on projecting pathologic evaluation beyond what is currently possible, perhaps globally.

In vivo assessment of human corneal epithelial cells in orthokeratology lens wearers: A pilot study.

Central corneal epithelial thinning associated with midperipheral epithelial thickening has been reported as the main factor contributing to the effectiveness of orthokeratology (ortho-k) in myopia control. Yet, the cellular mechanism governing the regional change in refractive power remains elusive. This study aimed to evaluate the correlation between the regional change in corneal epithelial thickness and cell density in ortho-k wearers. A new human prototype of a polarization-dependent optical coherence microscope was developed to enable noncontact and noninvasive in vivo imaging of corneal epithelial cells in ortho-k wearers with and without their ortho-k lens. The epithelial thickness and cell density were evaluated at the central and midperipheral corneal locations in four ortho-k wearers and four spectacle wearers serving as controls. Polarization-dependent optical coherence microscope achieved in vivo volumetric imaging of all epithelial cell types in ortho-k wearers with and without their lens over a field of view of 0.5 × 0.5 mm2 with an isotropic resolution of ~2.2 mm. The central epithelial thinning and midperipheral epithelial thickening were consistent across all ortho-k wearers. However, the inconsistency in their regional epithelial cell density highlighted a great variability in individual response to ortho-k treatment. There was no strong correlation between epithelial thickness and cell density, especially at the midperipheral cornea, in ortho-k participants. This study constitutes our first step toward uncovering the cellular mechanism underlying the effectiveness of ortho-k in myopia control. Future studies will focus on the longitudinal evaluation of epithelial cells before and during ortho-k treatment to identify factors governing individual response to ortho-k treatment and ultimately inform the dynamics of epithelial cells taking place during the ortho-k treatment.

Blood cell characterization based on deep learning and diffraction phase microscopy

Quantitative characterization of blood cells is very important for medical diagnosis and life science research. This study presents a novel label-free and high-throughput approach for analyzing flowing blood cells in a microfluidic channel by combining deep learning with partially coherent illumination-based diffraction phase microscopy (PCI-DPM). Diluted blood was injected into a microchannel and imaged with PCI-DPM, yielding high-contrast phase images for the blood cells. A modified YOLOv5-s neural network was used to perform automatic identification and analysis of blood cells in the phase image of PCI-DPM, revealing the diameter, dry mass, volumetric density, and speed of blood cells precisely. The proposed approach allows quick and accurate assessment of blood samples, providing valuable insights for patients in different pathological states, such as hematological disorders and infections.

Bichromatic tetraphasic full-field optical coherence microscopy.

Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast. We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts. BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast. BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors. BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.

Complex master-slave enhanced optical coherence microscopy

We present an instrument designed to facilitate localization and high-resolution, optical coherence microscopy (OCM) imaging of small biological samples immersed in a medium several orders of magnitude greater in volume. A modified turret-equipped microscope stand was inserted into the sample arm of a spectral domain optical coherence microscopy (SD-OCM) system. The instrument enabled swift change of imaging objectives through the incorporation of complex master-slave interferometry (CMSI), providing tolerance to dispersion for any objective through the acquisition of a few (≥2) calibration spectra. We demonstrate the instrument’s ability to localize and image samples by providing examples of its application to optical phantoms and to a porcine oocyte immersed in a biological culture medium.

The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Spindle shape and volume differ in high- and low-quality metaphase II oocytes.

Optical coherence microscopy non-invasively visualizes metaphase II spindles allowing for quantitative analysis of their volume and shape, which may prove useful in the assessment of the oocyte quality. Using a mouse model, we showed also that analysis of spindle length combined with morphokinetics improves the evaluation of the resulting embryos. The proper development of embryos strongly depends on the quality of oocytes, so the evaluation of oocytes may be a useful initial step in IVF procedures. Additionally, it enables embryologists to make more informed decisions regarding the treatments chosen for the patients and better manage patients' expectations. Optical coherence microscopy (OCM) allows for non-invasive 3D visualization of intracellular structures, such as spindles or nuclei, which have been linked to the success of embryonic development. Here, we applied a mouse model to examine whether OCM imaging could be used in the quality assessment of metaphase II (MII) oocytes. We showed that quantitative parameters describing the shape and volume of the MII spindle were associated with the quality of the resulting embryos, including the likelihood of blastocyst formation and the embryos' ability to differentiate the trophectoderm and primitive endoderm, but not the epiblast. We also created a multivariate linear regression model, combining OCM-based quantification of MII spindles with morphokinetic analysis of the embryos, that allowed for improved evaluation of the embryo quality. Finally, we proved that OCM does not interfere with the viability of the scanned cells, at least during the preimplantation development. Therefore, we believe that OCM-based quantitative assessment of MII spindles can improve the oocyte and embryo selection in IVF procedures.

Assessing cell viability with dynamic optical coherence microscopy.

Assessing cell viability is important in many fields of research. Current optical methods to assess cell viability typically involve fluorescent dyes, which are often less reliable and have poor permeability in primary tissues. Dynamic optical coherence microscopy (dOCM) is an emerging tool that provides label-free contrast reflecting changes in cellular metabolism. In this work, we compare the live contrast obtained from dOCM to viability dyes, and for the first time to our knowledge, demonstrate that dOCM can distinguish live cells from dead cells in murine syngeneic tumors. We further demonstrate a strong correlation between dOCM live contrast and optical redox ratio by metabolic imaging in primary mouse liver tissue. The dOCM technique opens a new avenue to apply label-free imaging to assess the effects of immuno-oncology agents, targeted therapies, chemotherapy, and cell therapies using live tumor tissues.