Full-field Optical Coherence Microscopy Research Articles

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.

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.

Advancing full-field metrology: rapid 3D imaging with geometric phase ferroelectric liquid crystal technology in full-field optical coherence microscopy.

Optical coherence microscopy (OCM) is a variant of OCT in which a high-numerical aperture lens is used. Full-field OCM (FF-OCM) is an emerging non-invasive, label-free, interferometric technique for imaging of surface structures or semi-transparent biomedical subjects with micron-scale resolutions. Different approaches to three dimensional full-field optical metrology are reviewed. The usual method for the phase-shifting technique in FF-OCM involves mechanically moving a mirror to change the optical path difference for obtaining en-face OCM images. However, with the use of a broadband source in FF-OCM, the phase shifts of different spectral components are not the same, resulting in the ambiguities in 3D image reconstruction. In this study, we demonstrate, by imaging tissues and cells, a unique geometric phase-shifter based on ferroelectric liquid crystal technology, to realize achromatic phase-shifting for rapid three-dimensional imaging in a FF-OCM system.

Full-field Optical Coherence Microscopy for Histology-like Analysis of Stromal Features in Corneal Grafts.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

Label-free, non-invasive, and repeatable cell viability bioassay using dynamic full-field optical coherence microscopy and supervised machine learning.

We present a novel method that can assay cellular viability in real-time using supervised machine learning and intracellular dynamic activity data that is acquired in a label-free, non-invasive, and non-destructive manner. Cell viability can be an indicator for cytology, treatment, and diagnosis of diseases. We applied four supervised machine learning models on the observed data and compared the results with a trypan blue assay. The cell death assay performance by the four supervised models had a balanced accuracy of 93.92 ± 0.86%. Unlike staining techniques, where criteria for determining viability of cells is unclear, cell viability assessment using machine learning could be clearly quantified.

Ultra-parallel label-free optophysiology of neural activity

SummaryThe electrical activity of neurons has a spatiotemporal footprint that spans three orders of magnitude. Traditional electrophysiology lacks the spatial throughput to image the activity of an entire neural network; besides, labeled optical imaging using voltage-sensitive dyes and tracking Ca2+ ion dynamics lack the versatility and speed to capture fast-spiking activity, respectively. We present a label-free optical imaging technique to image the changes to the optical path length and the local birefringence caused by neural activity, at 4,000 Hz, across a 200 × 200 μm2 region, and with micron-scale spatial resolution and 300-pm displacement sensitivity using Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM). The undulations in the optical responses from mammalian neuronal activity were matched with field-potential electrophysiology measurements and validated with channel blockers. By directly tracking the widefield neural activity at millisecond timescales and micrometer resolution, SPoOF OCM provides a framework to progress from low-throughput electrophysiology to high-throughput ultra-parallel label-free optophysiology.

Quantitative evaluation of the dynamic activity of HeLa cells in different viability states using dynamic full-field optical coherence microscopy

Dynamic full-field optical coherence microscopy (DFFOCM) was used to characterize the intracellular dynamic activities and cytoskeleton of HeLa cells in different viability states. HeLa cell samples were continuously monitored for 24 hours and compared with histological examination to confirm the cell viability states. The averaged mean frequency and magnitude observed in healthy cells were 4.79±0.5 Hz and 2.44±1.06, respectively. In dead cells, the averaged mean frequency was shifted to 8.57±0.71 Hz, whereas the magnitude was significantly decreased to 0.53±0.25. This cell dynamic activity analysis using DFFOCM is expected to replace conventional time-consuming and biopsies-required histological or biochemical methods.

Simultaneous 4-phase-shifted full-field optical coherence microscopy.

A new method is presented for full-field optical coherence tomography imaging, which permits capturing single shot phase sensitive imaging through simultaneous acquisition of four phase-shifted images with a single camera using unpolarized light for object illumination. Our method retains the full dynamic range of the camera by using different areas of a single camera sensor to capture each image. We demonstrate the performance of our method by imaging phantoms and live cultures of fibroblast, cancer, and macrophage cells to achieve 59 dB sensitivity with isotropic resolution down to 1 μm, and displacement sensitivity down to 0.1 nm. Our method can serve as a platform for developing high resolution imaging systems because when used in conjunction with broadband spatially incoherent light sources, the resolution is not affected by optical aberrations or speckle noise.

Differential phase imaging in full-field optical coherence microscopy using a short multimode fiber probe.

We demonstrate phase imaging that reduces the common phase noise in full-field optical coherence microscopy using a short multimode fiber (SMMF) probe. Using a cover glass, phase images of the SMMF and sample surfaces were measured simultaneously. Subtracting the phase of the SMMF surface as a reference, the phase drifts in the sample region are reduced. The axial and lateral resolutions were 2.3 µm and <4.4µm, respectively. The standard deviation of the time variation in the phase decreased from 14.3 deg to 9.2 deg and was reduced by 64% when in contact with the polymer film at the SMMF. In quantitative evaluations, the measured phases closely correspond to the phases changed by a piezoelectric device.

Processing conditions using electric discharge and optical characteristics for a short multimode fiber imaging probe

Using a short multimode fiber (SMMF) imaging probe with a diameter of 125μm and a length of 7.4 mm, the full-field optical coherence microscopy (FF OCM) to measure sectional images of in vivo rat brains deeper than 4 mm have been reported (Sato, 2019). However, since the tip of the SMMF was as cut, the process should be studied for smooth insertions into tissues and appropriate contacts to tissues. The SMMF processing conditions using electric discharge and the optical characteristics of the processed SMMF were investigated. Using a high voltage of 12,000 V for discharge duration of 11 s, the fiber tips of SMMF were processed to fabricate a spherical shape with a 77μm curvature radius. The measured imaging conditions approximate the optical model calculations of the parabolic refractive index and the spherical surface. Local blur and distortions in the measured images were identified in detail. Using the processed SMMF with a length of 7.35 mm and a diameter of 125μm, the transmission image of a chicken tendon samples closely corresponded to the image recorded with an optical microscope.

Corneal stromal stem cells restore transparency after N2 injury in mice.

Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N2‐injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen.

In Vivo Rat Brain Imaging through Full-Field Optical Coherence Microscopy Using an Ultrathin Short Multimode Fiber Probe

We demonstrate full-field optical coherence microscopy (OCM) using an ultrathin forward-imaging short multimode fiber (SMMF) probe with a core diameter of 50 μm, outer diameter of 125 μm, and length of 7.4 mm, which is a typical graded-index multimode fiber used for optical communications. The axial and lateral resolutions were measured to be 2.14 μm and 2.3 μm, respectively. By inserting the SMMF 4 mm into the cortex of an in vivo rat brain, scanning was performed to a depth of 147 μm from the SMMF facet with a field of view of 47 μm. Three-dimensional (3D) OCM images were obtained at depths ranging from approximately 20 μm to 90 μm. Based on the morphological information of the resliced 3D images and the dependence of the integration of the OCM image signal on the insertion length, the obtained 3D information of nerve fibers has been presented.

A compact high-speed full-field optical coherence microscope for high-resolution in vivo skin imaging.

A compact high-speed full-field optical coherence microscope has been developed for high-resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm3 and a weight of 210 g. Full-field illumination with low-coherence light is achieved with a high-brightness broadband light-emitting diode. High-speed full-field detection is achieved by using part of the image sensor of a high-dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm2 . Images of human skin, revealing in-depth cellular-level structures, were obtained in vivo and in real-time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm2 , but at a reduced depth.

A Feasibility Study of Broadband White Light Emitting Diode (WLED) Based Full-Field Optical Coherence Microscopy (FF-OCM) Using Derivative-Based Algorithm

Developing cost-effective and fast detection methods in full-field optical coherence microscopy (FF-OCM) platform for biological applications is of prime focus today. In this paper, we report a cost-effective Linnik type FF-OCM system with a single broadband white light emitting diode (WLED) (470–850 nm, λ0 = 630 nm) light source in conjunction with a derivative-based image reconstruction algorithm. Sequential 2-D multiple phase-shifted interferograms were obtained by moving the sample stage using piezoelectric transducer and recorded via a 2-D charge complementary oxide semiconductor camera. Subsequently, the en-face sectional images were reconstructed using a derivative-based algorithm. The measured axial and lateral resolutions along with interferometric phase stability of the system were 0.9 μm, 1.4 μm, and 11 mrad, respectively. The advantages of the derivative-based algorithm over 4-step phase shifted method were demonstrated using USAF target and onion as test samples. Moreover, studies via the present system of the microstructure of skin epidermis in mouse ear with melanoma showed a significant deterioration from normal mouse skin in stratum corneum of the epidermis. This is the first study of the application of a derivative-based algorithm for biological samples in FF-OCM wherein WLED is used as a light source. The present system is stable, compact, and cost-effective compared to the conventional FF-OCM systems and provides comparable spatial resolution.

Focus defect and dispersion mismatch in full-field optical coherence microscopy.

Full-field optical coherence microscopy (FFOCM) is an optical technique, based on low-coherence interference microscopy, for tomographic imaging of semi-transparent samples with micrometer-scale spatial resolution. The differences in refractive index between the sample and the immersion medium of the microscope objectives may degrade the FFOCM image quality because of focus defect and optical dispersion mismatch. These phenomena and their consequences are discussed in this theoretical paper. Experimental methods that have been implemented in FFOCM to minimize the adverse effects of these phenomena are summarized and compared.

New parameters in assessment of human donor corneal stroma.

To provide quantitative parameters for assessment of human donor corneal stroma by imaging stromal features of diseased and normal human corneas with full-field optical coherence microscopy (FFOCM), using confocal microscopy (CM) and histology as reference techniques. Bowman's layer (BL) thickness and keratocyte density were assessed exvivo in 23 human donor corneas and 27 human pathological corneas (keratoconus and other corneal disorders) with FFOCM, CM and histology. Stromal backscattering was assessed with FFOCM. Additionally, 10 normal human corneas were assessed invivo with CM. In FFOCM, the logarithm of the normalized stromal reflectivity was a linear function of stromal depth (R2 =0.95) in human donor corneas. Compared with keratoconus corneas, human donor corneas featured higher BL thickness (p=0.0014) with lower coefficient of variation (BL-COV; p=0.0002), and linear logarithmic stromal reflectivity with depth (higher R2 , p=0.0001). Compared with other corneal disorders, human donor corneas featured lower BL-COV (p=0.012) and higher R2 (p=0.0001). Using the 95% confidence limits of the human donor cornea group, BL thickness<6.5μm (sensitivity, 57%; specificity, 100%), BL-COV>18.6% (79%; 100%) and R2 <0.94 (93%; 71%) were revealed as indictors of abnormal cornea. In CM, keratocyte density decreased with stromal depth (r=-0.56). The mean overall keratocyte density (cells/mm2 ) was 205 in human donor corneas, 244 in keratoconus, 176 in other corneal disorders and 386 in normal corneas. Full-field optical coherence microscopy (FFOCM) provides precise and reliable parameters for non-invasive assessment of human donor corneal stroma during storage, enabling detection of stromal disorders that could impair the results of keratoplasty.

En face coherence microscopy [Invited

En face coherence microscopy or flying spot or full field optical coherence tomography or microscopy (FF-OCT/FF-OCM) belongs to the OCT family because the sectioning ability is mostly linked to the source coherence length. In this article we will focus our attention on the advantages and the drawbacks of the following approaches: en face versus B scan tomography in terms of resolution, coherent versus incoherent illumination and influence of aberrations, and scanning versus full field imaging. We then show some examples to illustrate the diverse applications of en face coherent microscopy and show that endogenous or exogenous contrasts can add valuable information to the standard morphological image. To conclude we discuss a few domains that appear promising for future development of en face coherence microscopy.

Calculation of the interference pattern formed by image-carrying light beams diffracted by an acoustic wave in a uniaxial crystal

A scheme of full-field optical coherence microscopy based on a Michelson interferometer with wideband light source and acousto-optic spectral filtration in the output channel is studied. Mathematical formulas for calculating the interference pattern formed by image-carrying light beams diffracted by an acoustic wave in a uniaxial crystal are derived for this scheme. Results of numerical modeling and its comparison with experimental data are given.

Imaging Microscopic Features of Keratoconic Corneal Morphology.

Purpose:To search for gold-standard histology indicators using alternative imaging modalities in keratoconic corneas.Methods:Prospective observational case–control study. Fourteen keratoconic corneas and 20 normal corneas (10 in vivo healthy subjects and 10 ex vivo donor corneas) were examined. Images of corneas were taken by spectral domain optical coherence tomography (SD-OCT) and in vivo confocal microscopy (IVCM) before keratoplasty. The same removed corneal buttons were imaged after keratoplasty with full-field optical coherence microscopy (FFOCM) and then fixed and sent for histology. Controls consisted of normal subjects imaged in vivo with IVCM and donor corneas imaged ex vivo with FFOCM. Corneal structural changes related to pathology were noted with each imaging modality. Cell density was quantified by manual cell counting.Results:Keratoconus indicators (ie, epithelial thinning/thickening, cell shape changes, ferritin deposits, basement membrane anomalies, Bowman layer thinning, ruptures, interruptions, scarring, stromal modifications, and appearance of Vogt striae) were generally visible with all modalities. Additional features could be seen with FFOCM in comparison with gold-standard histology, particularly in the Bowman layer region, whereas the combination of SD-OCT plus IVCM detected 76% of those features detected in histology. Three-dimensional FFOCM imaging aided interpretation of two-dimensional IVCM and SD-OCT data. Basal epithelial cell and keratocyte densities were significantly lower in patients with keratoconus than those in normals (P < 0.0001).Conclusions:Structural and cellular assessment of the keratoconic cornea by means of either in vivo SD-OCT combined with IVCM or ex vivo FFOCM in both cross-sectional and en face views can detect as many keratoconus indicators as gold-standard histology.

Ultrathin forward-imaging short multimode fiber probe for full-field optical coherence microscopy

To extend the applications of optical coherence tomography (OCT) to the fields of physiology and clinical medicine, less invasive, robust, and reliable optical probes are required. Thus, we demonstrate an ultrathin forward-imaging short multimode fiber (SMMF) optical coherence microscopy (OCM) probe with a 50 μm core diameter, 125μm total diameter, and 5.12mm length. Imaging conditions and magnification were analyzed, and they correspond closely to the measured results. The dispersion of the SMMF was investigated, and the modal dispersion coefficient was found to be 2.3% of the material dispersion coefficient. The axial resolution was minimized at 2.15μm using a 0.885-mm-thick dispersion compensator. The lateral resolution was evaluated to be 4.38μm using a test pattern. The contrast of the OCM images was 5.7 times higher than that of the signal images owing to the coherence gate. The depth of focus and diameter of the field of view were measured to be 60μm and 40–50 μm, respectively. OCM images of the dried fins of small fish (Medaka) were measured and internal structures could be recognized.