Recovery of angular scattering profiles through a flexible multimode fiber.

Endoscopic angle-resolved light scattering methods have been developed for early cancer detection but they typically require multi-element coherent fiber optic bundles to recover scattering distributions from tissues. Recent work has focused on using a single multimode fiber (MMF) to measure angle resolved scattering but this approach has practical limitations to overcome before clinical translation. Here we address these limitations by proposing an MMF-based endoscope capable of measuring angular scattering patterns suitable for determining structure. Significantly, this approach implements a spectrally resolved detection scheme to reduce speckle and leverages the azimuthal symmetry of the angular scattering patterns to enable measurements that are robust to fiber bending. This results in a unique method that does not require matrix inversion or machine learning to measure a transmitted scattering distribution. The MMF utilized here is 1000 mm in length with a 200 µm core and is demonstrated to recover angular scattering distributions even with bending displacements of up to 30 cm. This advance has a significant impact on the clinical translation of biomedical endoscopic diagnostic techniques that use angular scattering to determine the size of cell nuclei to detect early cancer.

Utilizing quantitative phase microscopy to localize fluorescence in three dimensions via the transport of intensity equation.

We demonstrate the use of a novel, to the best of our knowledge, localization algorithm for digitally refocusing fluorescence images from a three-dimensional cell culture. Simultaneous phase and fluorescence intensity images are collected through a multimodal system that combines digital holography via quantitative phase microscopy (QPM) and fluorescence microscopy. Defocused fluorescence images are localized to a specific z-plane within the three-dimensional (3D) matrix using the transport of intensity equation (TIE) and depth-resolved information derived from the QPM measurements. This technique is applied to cells stained with different fluorescent tags suspended in 3D collagen hydrogel cultures. Experimental findings demonstrate the localization of defocused images, facilitating the analysis and comparison of cells within the hydrogel matrix. This method holds promise for comprehensive cellular imaging of fluorescence labeling in three-dimensional environments, enabling detailed investigations into cellular behavior and interactions.

Multiscale optical phase fluctuations link disorder strength and fractal dimension of cell structure.

Optical methods for examining cellular structure based on endogenous contrast rely on analysis of refractive index changes to discriminate cell phenotype. These changes can be visualized using techniques such as phase contrast microscopy, detected by light scattering, or analyzed numerically using quantitative phase imaging. The statistical variations of refractive index at the nanoscale can be quantified using disorder strength, a metric seen to increase with neoplastic change. In contrast, the spatial organization of these variations is typically characterized using a fractal dimension, which is also seen to increase with cancer progression. Here, we seek to link these two measurements using multiscale measurements of optical phase to calculate disorder strength and in turn to determine the fractal dimension of the structures. First, quantitative phase images are analyzed to show that the disorder strength metric changes with resolution. The trend of disorder strength with length scales is analyzed to determine the fractal dimension of the cellular structures. Comparison of these metrics is presented for different cell lines with varying phenotypes including MCF10A, MCF7, BT474, HT-29, A431, and A549 cell lines, in addition to three cell populations with modified phenotypes. Our results show that disorder strength and fractal dimension can both be obtained with quantitative phase imaging and that these metrics can independently distinguish between different cell lines. Furthermore, their combined use presents a new approach for better understanding cellular restructuring during different pathways.

Biophysical Profiling of Sickle Cell Disease Using Holographic Cytometry and Deep Learning.

Sickle cell disease (SCD) is an inherited hematological disorder associated with high mortality rates, particularly in sub-Saharan Africa. SCD arises due to the polymerization of sickle hemoglobin, which reduces flexibility of red blood cells (RBCs), causing blood vessel occlusion and leading to severe morbidity and early mortality rates if untreated. While sickle solubility tests are available to sub-Saharan African population as a means for detecting sickle hemoglobin (HbS), the test falls short in assessing the severity of the disease and visualizing the degree of cellular deformation. Here, we propose use of holographic cytometry (HC), a high throughput, label-free imaging modality, for comprehensive morphological profiling of RBCs as a means to detect SCD. For this study, more than 2.5 million single-cell holographic images from normal and SCD patient samples were collected using the HC system. We have developed an approach for specially defining training data to improve machine learning classification. Here, we demonstrate the deep learning classifier developed using this approach can produce highly accurate classification, even on unknown patient samples.

Esophageal OCT Imaging Using a Paddle Probe Externally Attached to Endoscope.

BACKGROUND AND AIMS: Endoscopic surveillance of Barrett's esophagus (BE) by white light examination is insufficient to diagnose dysplastic change. In this work, we describe an optical imaging method to obtain high-resolution cross-sectional imaging using a paddle-shaped probe affixed to the endoscope tip. METHODS: We integrated Optical Coherence Tomography (OCT), an optical imaging method that produces cross-sectional images, into a paddle probe attached to video endoscope. We acquired images of esophageal epithelium from patients undergoing routine upper GI endoscopy. Images were classified by a reviewer blinded to patient identity and condition, and these results were compared with clinical diagnosis. RESULTS: We successfully captured epithelial OCT images from 30 patients and identified features consistent with both squamous epithelium and Barrett's esophagus. Our blinded image reviewer classified BE versus non-BE with 91.5% accuracy (65/71 image regions), including sensitivity of 84.6% for BE (11/13) and a specificity of 93.1% (54/58). However, in 16 patients, intubation of the probe into the esophagus could not be achieved. CONCLUSIONS: A paddle probe is a feasible imaging format for acquiring cross-sectional OCT images from the esophagus and can provide a structural assessment of BE and non-BE tissue. Probe form factor is the current limiting obstacle, but could be addressed by further miniaturization.

Shear Modulus Measurement by Quantitative Phase Imaging and Correlation with Atomic Force Microscopy.

Many approaches have been developed to characterize cell elasticity. Among these, atomic force microscopy (AFM) combined with modeling has been widely used to characterize cellular compliance. However, such approaches are often limited by the difficulties associated with using a specific instrument and by the complexity of analyzing the measured data. More recently, quantitative phase imaging (QPI) has been applied to characterize cellular stiffness by using an effective spring constant. This metric was further correlated to mass distribution (disorder strength) within the cell. However, these measurements are difficult to compare to AFM-derived measurements of Young's modulus. Here, we describe, to our knowledge, a new way of analyzing QPI data to directly retrieve the shear modulus. Our approach enables label-free measurement of cellular mechanical properties that can be directly compared to values obtained from other rheological methods. To demonstrate the technique, we measured shear modulus and phase disorder strength using QPI, as well as Young's modulus using AFM, across two breast cancer cell-line populations dosed with three different concentrations of cytochalasin D, an actin-depolymerizing toxin. Comparison of QPI-derived and AFM moduli shows good agreement between the two measures and further agrees with theory. Our results suggest that QPI is a powerful tool for cellular biophysics because it allows for optical quantitative measurements of cell mechanical properties.

Optical coherence tomography through a rigid borescope applied to quantification of articular cartilage thickness in a porcine knee model.

There exists an unmet need for an optical coherence tomography (OCT) delivery scheme that is simple, robust, and applicable to general surgical applications. To deliver the beam in a narrow form factor, optical borescopes present an attractive potential solution. We present a method for enabling endoscopic delivery of OCT using a handheld rigid borescope adapted to a low-cost OCT engine. The system reduces the distal profile of the scanner, enabling application of the system in otherwise hard-to-access regions. The clinical potential of this design is demonstrated through real-time quantification of articular cartilage thickness, a primary biomarker of joint health during osteoarthritis. This platform has the potential to enable use of OCT for real-time feedback during arthroscopic surgery.

Cellular shear stiffness reflects progression of arsenic-induced transformation during G1.

Cancer cells consistently exhibit decreased stiffness; however, the onset and progression of this change have not been characterized. To study the development of cell stiffness changes, we evaluated the shear stiffness of populations of cells during transformation to a carcinogenic state. Bronchial epithelial cells were exposed to sodium arsenite to initiate early stages of transformation. Exposed cells were cultured in soft agar to further transformation and select for clonal populations exhibiting anchorage-independent growth. Shear stiffness of various cell populations in G1 was assessed using a novel non-invasive assay that applies shear stress with fluid flow and evaluates nanoscale deformation using quantitative phase imaging (QPI). Arsenic-treated cells exhibited reduced stiffness relative to control cells, while arsenic clonal lines, selected by growth in soft agar, were found to have reduced stiffness relative to control clonal lines, which were cultured in soft agar but did not receive arsenic treatment. The relative standard deviation (RSD) of the stiffness of Arsenic clones was reduced compared with control clones, as well as to the arsenic-exposed cell population. Cell stiffness at the population level exhibits potential to be a novel and sensitive framework for identifying the development of cancerous cells.

Comparison of imaging fiber bundles for coherence-domain imaging.

Use of imaging fiber bundles for coherence-domain imaging has remained limited to date. In this work, we provide characterization of commercially available imaging bundles for coherence-domain imaging, by evaluating their modal structure for applicability to interferometric imaging. We further examine custom fabricated bundles developed in collaboration with a corporate partner for their ability to reduce interelement optical path length variability and cross talk between elements. The results presented here will serve as a useful guide for comparing fiber bundles for coherence imaging while also offering an improved understanding of the functionality and limitations of imaging bundles for advancing coherent imaging technologies.

Optical Phase Measurements of Disorder Strength Link Microstructure to Cell Stiffness.

There have been sustained efforts on the part of cell biologists to understand the mechanisms by which cells respond to mechanical stimuli. To this end, many rheological tools have been developed to characterize cellular stiffness. However, measurement of cellular viscoelastic properties has been limited in scope by the nature of most microrheological methods, which require direct mechanical contact, applied at the single-cell level. In this article, we describe, to our knowledge, a new analysis approach for quantitative phase imaging that relates refractive index variance to disorder strength, a parameter that is linked to cell stiffness. Significantly, both disorder strength and cell stiffness are measured with the same phase imaging system, presenting a unique alternative for label-free, noncontact, single-shot imaging of cellular rheologic properties. To demonstrate the potential applicability of the technique, we measure phase disorder strength and shear stiffness across five cellular populations with varying mechanical properties and demonstrate an inverse relationship between these two parameters. The existence of this relationship suggests that predictions of cell mechanical properties can be obtained from examining the disorder strength of cell structure using this, to our knowledge, novel, noncontact technique.

Feasibility of clinical detection of cervical dysplasia using angle-resolved low coherence interferometry measurements of depth-resolved nuclear morphology.

This study sought to establish the feasibility of using in situ depth-resolved nuclear morphology measurements for detection of cervical dysplasia. Forty enrolled patients received routine cervical colposcopy with angle-resolved low coherence interferometry (a/LCI) measurements of nuclear morphology. a/LCI scans from 63 tissue sites were compared to histopathological analysis of co-registered biopsy specimens which were classified as benign, low-grade squamous intraepithelial lesion (LSIL), or high-grade squamous intraepithelial lesion (HSIL). Results were dichotomized as dysplastic (LSIL/HSIL) versus non-dysplastic and HSIL versus LSIL/benign to determine both accuracy and potential clinical utility of a/LCI nuclear morphology measurements. Analysis of a/LCI data was conducted using both traditional Mie theory based processing and a new hybrid algorithm that provides improved processing speed to ascertain the feasibility of real-time measurements. Analysis of depth-resolved nuclear morphology data revealed a/LCI was able to detect a significant increase in the nuclear diameter at the depth bin containing the basal layer of the epithelium for dysplastic versus non-dysplastic and HSIL versus LSIL/Benign biopsy sites (both p < 0.001). Both processing techniques resulted in high sensitivity and specificity (>0.80) in identifying dysplastic biopsies and HSIL. The hybrid algorithm demonstrated a threefold decrease in processing time at a slight cost in classification accuracy. The results demonstrate the feasibility of using a/LCI as an adjunctive clinical tool for detecting cervical dysplasia and guiding the identification of optimal biopsy sites. The faster speed from the hybrid algorithm offers a promising approach for real-time clinical analysis.

Scanning system for angle-resolved low-coherence interferometry.

Angle-resolved low-coherence interferometry (a/LCI) detects precancer by enabling depth-resolved measurements of nuclear morphology in vivo. A significant limitation of a/LCI is the point-probe nature of the method, sampling <0.5 mm2 before probe relocation is necessary. In this work, we demonstrate a scanning method capable of assessing an area >100 mm2 without repositioning. By utilizing a reflection-only three-optic rotator prism and a two-axis scanning mirror, we demonstrate radial scans of a sample with a linear range of 12 mm and a full rotational range of 180°. Use of this design will improve the diagnostic utility of a/LCI for wide-area screening of tissue health.

Dual-axis optical coherence tomography for deep tissue imaging.

We have developed dual-axis optical coherence tomography (DA-OCT) which enables deep tissue imaging by using a novel off-axis illumination/detection configuration. DA-OCT offers a 100-fold speed increase compared with its predecessor, multispectral multiple-scattering low coherence interferometry (ms2/LCI), by using a new beam scanning mechanism based on a microelectro-mechanical system (MEMS) mirror. The data acquisition scheme was altered to take advantage of this scanning speed, producing tomographic images at a rate of 4 frames (B-scans) per second. DA-OCT differs from ms2/LCI in that the dual axes intersect at a shallower depth (∼1 mm). This difference, coupled with the faster scanning speed, shifts the detection priority from multiply scattered to ballistic light. The utility of this approach was demonstrated by imaging both ex vivo porcine ear skin and in vivo rat skin from a McFarlane flap model. The enhanced penetration depth provided by the DA-OCT system will be beneficial to various clinical applications in dermatology and surgery.

Imaging deformation of adherent cells due to shear stress using quantitative phase imaging.

We present a platform for detecting cellular deformations from mechanical stimuli, such as fluid shear stress, using rapid quantitative phase imaging. Rapid quantitative phase imaging was used to analyze changes in the optical path length of adherent skin cancer cells during mechanical displacement. Both the whole-cell phase displacement and the resultant shift of the cellular center of mass were calculated over the duration of the stimulus. Whole-cell phase displacement images were found to match expectation. Furthermore, center-of-mass shifts of adherent cells were found to resemble that of a one-dimensional Kelvin-Voigt (KV) viscoelastic solid. Cellular steady-state displacements from step fluid shear stimuli were found to be linearly related to the shear stress. Shear stiffness constants for cells exposed to a cytoskeletal disrupting toxin were found to be significantly lower than unexposed cells. This novel technique allows for elastographic analysis of whole-cell effective shear stiffness without the use of an exogenous force applicator, a specialized culture substrate, or tracking net perimeter movement of the cell.

Spatial frequency-domain multiplexed microscopy for simultaneous, single-camera, one-shot, fluorescent, and quantitative-phase imaging.

Multimodal imaging is a crucial tool when imaging biological phenomena that cannot be comprehensively captured by a single modality. Here, we introduce a theoretical framework for spatial-frequency-multiplexed microscopy via off-axis interference as a novel wide-field imaging technique that enables true simultaneous multimodal and multichannel wide-field imaging. We experimentally demonstrate this technique for single-camera, simultaneous two-channel fluorescence and one-channel quantitative-phase imaging for fluorescent microspheres and fixed cells stained for F-actin and nuclear fluorescence.

Quantitative phase microscopy with off-axis optical coherence tomography.

We have developed a modality for quantitative phase imaging within spectral domain optical coherence tomography based on using an off-axis reference beam. By tilting the propagation of the reference beam relative to that of the sample beam, a spatially varying fringe is generated. Upon detection of this fringe using a parallel spectral domain scheme, the fringe can be used to separate the interference component of the signal and obtain the complex sample field. In addition to providing quantitative phase measurements within a depth resolved measurement, this approach also allows elimination of the complex conjugate artifact, a known limitation of spectral interferometry. The principle of the approach is described here along with demonstration of its capabilities using technical samples.

In vivo analysis of burns in a mouse model using spectroscopic optical coherence tomography.

Spectroscopic analysis of biological tissues can provide insight into changes in structure and function due to disease or injury. Depth-resolved spectroscopic measurements can be implemented for tissue imaging using optical coherence tomography (OCT). Here, spectroscopic OCT is applied to in vivo measurement of burn injury in a mouse model. Data processing and analysis methods are compared for their accuracy. Overall accuracy in classifying burned tissue was found to be as high as 91%, producing an area under the curve of a receiver operating characteristic curve of 0.97. The origins of the spectral changes are identified by correlation with histopathology.

Noise characterization of supercontinuum sources for low-coherence interferometry applications.

We examine the noise properties of supercontinuum light sources when used in low-coherence interferometry applications. The first application is a multiple-scattering low-coherence interferometry (ms2/LCI) system, where high power and long image acquisition times are required to image deep into tissue. For this system, we compare the noise characteristics of two supercontinuum sources from different suppliers. Both sources have long-term drift that limits the amount of time over which signal averaging is advantageous for reducing noise. The second application is a high-resolution optical coherence tomography system, where broadband light is needed for high axial resolution. For this system, we compare the noise performance of the two supercontinuum sources and a light source based on four superluminescent diodes (SLD) using imaging contrast as a comparative metric. We find that the NKT SuperK has superior noise performance compared with the Fianium SC-450-4, but neither meets the performance of the SLD.

Next-generation endoscopic probe for detection of esophageal dysplasia using combined OCT and angle-resolved low-coherence interferometry.

Angle-resolved low-coherence interferometry (a/LCI) is an optical technique that enables depth-specific measurements of nuclear morphology, with applications to detecting epithelial cancers in various organs. Previous a/LCI setups have been limited by costly fiber-optic components and large footprints. Here, we present a novel a/LCI instrument incorporating a channel for optical coherence tomography (OCT) to provide real-time image guidance. We showcase the system's capabilities by acquiring imaging data from in vivo Barrett's esophagus patients. The main innovation in this geometry lies in implementing a pathlength-matched single-mode fiber array, offering substantial cost savings while preserving signal fidelity. A further innovation is the introduction of a specialized side-viewing probe tailored for esophageal imaging, featuring miniature optics housed in a custom 3D-printed enclosure attached to the tip of the endoscope. The integration of OCT guidance enhances the precision of tissue targeting by providing real-time morphology imaging. This novel device represents a significant advancement in clinical translation of an enhanced screening approach for esophageal precancer, paving the way for more effective early-stage detection and intervention strategies.