Subcellular mechano-microscopy: high resolution three-dimensional elasticity mapping using optical coherence microscopy.

The importance of cellular-scale mechanical properties is well-established, yet it is challenging to map subcellular elasticity in three dimensions. We present subcellular mechano-microscopy, an optical coherence microscopy (OCM)-based variant of three-dimensional (3-D) compression optical coherence elastography (OCE) that provides an elasticity system resolution of 5 × 5 × 5 µm: a 7-fold improvement in system resolution over previous OCE studies of cells. The improved resolution is achieved through a ∼5-fold improvement in optical resolution, refinement of the strain estimation algorithm, and demonstration that mechanical deformation of subcellular features provides feature resolution far greater than that demonstrated previously on larger features with diameter >250 µm. We use mechano-microscopy to image adipose-derived stem cells encapsulated in gelatin methacryloyl. We compare our results with compression OCE and demonstrate that mechano-microscopy can provide contrast from subcellular features not visible using OCE.

Speckle-dependent accuracy in phase-sensitive optical coherence tomography.

Phase-sensitive optical coherence tomography (OCT) is used to measure motion in a range of techniques, such as Doppler OCT and optical coherence elastography (OCE). In phase-sensitive OCT, motion is typically estimated using a model of the OCT signal derived from a single reflector. However, this approach is not representative of turbid samples, such as tissue, which exhibit speckle. In this study, for the first time, we demonstrate, through theory and experiment that speckle significantly lowers the accuracy of phase-sensitive OCT in a manner not accounted for by the OCT signal-to-noise ratio (SNR). We describe how the inaccuracy in speckle reduces phase difference sensitivity and introduce a new metric, speckle brightness, to quantify the amount of constructive interference at a given location in an OCT image. Experimental measurements show an almost three-fold degradation in sensitivity between regions of high and low speckle brightness at a constant OCT SNR. Finally, we apply these new results in compression OCE to demonstrate a ten-fold improvement in strain sensitivity, and a five-fold improvement in contrast-to-noise by incorporating independent speckle realizations. Our results show that speckle introduces a limit to the accuracy of phase-sensitive OCT and that speckle brightness should be considered to avoid erroneous interpretation of experimental data.

Depth-encoded optical coherence elastography for simultaneous volumetric imaging of two tissue faces.

Depth-encoded optical coherence elastography (OCE) enables simultaneous acquisition of two three-dimensional (3D) elastograms from opposite sides of a sample. By the choice of suitable path-length differences in each of two interferometers, the detected carrier frequencies are separated, allowing depth-ranging from each interferometer to be performed simultaneously using a single spectrometer. We demonstrate depth-encoded OCE on a silicone phantom and a freshly excised sample of mouse liver. This technique minimizes the required spectral detection hardware and halves the total scan time. Depth-encoded OCE may expedite clinical translation in time-sensitive applications requiring rapid 3D imaging of multiple tissue surfaces, such as tumor margin assessment in breast-conserving surgery.

Investigation of optical coherence micro-elastography as a method to visualize micro-architecture in human axillary lymph nodes.

BACKGROUND: Evaluation of lymph node involvement is an important factor in detecting metastasis and deciding whether to perform axillary lymph node dissection (ALND) in breast cancer surgery. As ALND is associated with potentially severe long term morbidity, the accuracy of lymph node assessment is imperative in avoiding unnecessary ALND. The mechanical properties of malignant lymph nodes are often distinct from those of normal nodes. A method to image the micro-scale mechanical properties of lymph nodes could, thus, provide diagnostic information to aid in the assessment of lymph node involvement in metastatic cancer. In this study, we scan axillary lymph nodes, freshly excised from breast cancer patients, with optical coherence micro-elastography (OCME), a method of imaging micro-scale mechanical strain, to assess its potential for the intraoperative assessment of lymph node involvement. METHODS: Twenty-six fresh, unstained lymph nodes were imaged from 15 patients undergoing mastectomy or breast-conserving surgery with axillary clearance. Lymph node specimens were bisected to allow imaging of the internal face of each node. Co-located OCME and optical coherence tomography (OCT) scans were taken of each sample, and the results compared to standard post-operative hematoxylin-and-eosin-stained histology. RESULTS: The optical backscattering signal provided by OCT alone may not provide reliable differentiation by inspection between benign and malignant lymphoid tissue. Alternatively, OCME highlights local changes in tissue strain that correspond to malignancy and are distinct from strain patterns in benign lymphoid tissue. The mechanical contrast provided by OCME complements the optical contrast provided by OCT and aids in the differentiation of malignant tumor from uninvolved lymphoid tissue. CONCLUSION: The combination of OCME and OCT images represents a promising method for the identification of malignant lymphoid tissue. This method shows potential to provide intraoperative assessment of lymph node involvement, thus, preventing unnecessary removal of uninvolved tissues and improving patient outcomes.

Optical palpation: optical coherence tomography-based tactile imaging using a compliant sensor.

We present optical palpation, a tactile imaging technique for mapping micrometer- to millimeter-scale mechanical variations in soft tissue. In optical palpation, a stress sensor consisting of translucent, compliant silicone with known stress-strain behavior is placed on the tissue surface and a compressive load is applied. Optical coherence tomography (OCT) is used to measure the local strain in the sensor, from which the local stress at the sample surface is calculated and mapped onto an image. We present results in tissue-mimicking phantoms, demonstrating the detection of a feature embedded 4.7 mm below the sample surface, well beyond the depth range of OCT. We demonstrate the use of optical palpation to delineate the boundary of a region of tumor in freshly excised human breast tissue, validated against histopathology.

Multi-class classification of breast tissue using optical coherence tomography and attenuation imaging combined via deep learning.

We demonstrate a convolutional neural network (CNN) for multi-class breast tissue classification as adipose tissue, benign dense tissue, or malignant tissue, using multi-channel optical coherence tomography (OCT) and attenuation images, and a novel Matthews correlation coefficient (MCC)-based loss function that correlates more strongly with performance metrics than the commonly used cross-entropy loss. We hypothesized that using multi-channel images would increase tumor detection performance compared to using OCT alone. 5,804 images from 29 patients were used to fine-tune a pre-trained ResNet-18 network. Adding attenuation images to OCT images yields statistically significant improvements in several performance metrics, including benign dense tissue sensitivity (68.0% versus 59.6%), malignant tissue positive predictive value (PPV) (79.4% versus 75.5%), and total accuracy (85.4% versus 83.3%), indicating that the additional contrast from attenuation imaging is most beneficial for distinguishing between benign dense tissue and malignant tissue.

Quantitative Micro-Elastography Enables In Vivo Detection of Residual Cancer in the Surgical Cavity during Breast-Conserving Surgery.

Breast-conserving surgery (BCS) is commonly used for the treatment of early-stage breast cancer. Following BCS, approximately 20% to 30% of patients require reexcision because postoperative histopathology identifies cancer in the surgical margins of the excised specimen. Quantitative micro-elastography (QME) is an imaging technique that maps microscale tissue stiffness and has demonstrated a high diagnostic accuracy (96%) in detecting cancer in specimens excised during surgery. However, current QME methods, in common with most proposed intraoperative solutions, cannot image cancer directly in the patient, making their translation to clinical use challenging. In this proof-of-concept study, we aimed to determine whether a handheld QME probe, designed to interrogate the surgical cavity, can detect residual cancer directly in the breast cavity in vivo during BCS. In a first-in-human study, 21 BCS patients were scanned in vivo with the QME probe by five surgeons. For validation, protocols were developed to coregister in vivo QME with postoperative histopathology of the resected tissue to assess the capability of QME to identify residual cancer. In four cavity aspects presenting cancer and 21 cavity aspects presenting benign tissue, QME detected elevated stiffness in all four cancer cases, in contrast to low stiffness observed in 19 of the 21 benign cases. The results indicate that in vivo QME can identify residual cancer by directly imaging the surgical cavity, potentially providing a reliable intraoperative solution that can enable more complete cancer excision during BCS. SIGNIFICANCE: Optical imaging of microscale tissue stiffness enables the detection of residual breast cancer directly in the surgical cavity during breast-conserving surgery, which could potentially contribute to more complete cancer excision.

Analysis of sensitivity in quantitative micro-elastography.

Quantitative micro-elastography (QME), a variant of compression optical coherence elastography (OCE), is a technique to image tissue elasticity on the microscale. QME has been proposed for a range of applications, most notably tumor margin assessment in breast-conserving surgery. However, QME sensitivity, a key imaging metric, has yet to be systematically analyzed. Consequently, it is difficult to optimize imaging performance and to assess the potential of QME in new application areas. To address this, we present a framework for analyzing sensitivity that incorporates the three main steps in QME image formation: mechanical deformation, its detection using optical coherence tomography (OCT), and signal processing used to estimate elasticity. Firstly, we present an analytical model of QME sensitivity, validated by experimental data, and demonstrate that sub-kPa elasticity sensitivity can be achieved in QME. Using silicone phantoms, we demonstrate that sensitivity is dependent on friction, OCT focus depth, and averaging methods in signal processing. For the first time, we show that whilst lubrication of layer improves accuracy by reducing surface friction, it reduces sensitivity due to the time-dependent effect of lubricant exudation from the layer boundaries resulting in increased friction. Furthermore, we demonstrate how signal processing in QME provides a trade-off between sensitivity and resolution that can be used to optimize imaging performance. We believe that our framework to analyze sensitivity can help to sustain the development of QME and, also, that it can be readily adapted to other OCE techniques.

Optical palpation for tumor margin assessment in breast-conserving surgery.

Intraoperative margin assessment is needed to reduce the re-excision rate of breast-conserving surgery. One possibility is optical palpation, a tactile imaging technique that maps stress (force applied across the tissue surface) as an indicator of tissue stiffness. Images (optical palpograms) are generated by compressing a transparent silicone layer on the tissue and measuring the layer deformation using optical coherence tomography (OCT). This paper reports, for the first time, the diagnostic accuracy of optical palpation in identifying tumor within 1 mm of the excised specimen boundary using an automated classifier. Optical palpograms from 154 regions of interest (ROIs) from 71 excised tumor specimens were obtained. An automated classifier was constructed to predict the ROI margin status by first choosing a circle diameter, then searching for a location within the ROI where the circle was ≥ 75% filled with high stress (indicating a positive margin). A range of circle diameters and stress thresholds, as well as the impact of filtering out non-dense tissue regions, were tested. Sensitivity and specificity were calculated by comparing the automated classifier results with the true margin status, determined from co-registered histology. 83.3% sensitivity and 86.2% specificity were achieved, compared to 69.0% sensitivity and 79.0% specificity obtained with OCT alone on the same dataset using human readers. Representative optical palpograms show that positive margins containing a range of cancer types tend to exhibit higher stress compared to negative margins. These results demonstrate the potential of optical palpation for margin assessment.

Three-dimensional mapping of the attenuation coefficient in optical coherence tomography to enhance breast tissue microarchitecture contrast.

Effective intraoperative tumor margin assessment is needed to reduce re-excision rates in breast-conserving surgery (BCS). Mapping the attenuation coefficient in optical coherence tomography (OCT) throughout a sample to create an image (attenuation imaging) is one promising approach. For the first time, three-dimensional OCT attenuation imaging of human breast tissue microarchitecture using a wide-field (up to ~45 × 45 × 3.5 mm) imaging system is demonstrated. Representative results from three mastectomy and one BCS specimen (from 31 specimens) are presented with co-registered postoperative histology. Attenuation imaging is shown to provide substantially improved contrast over OCT, delineating nuanced features within tumors (including necrosis and variations in tumor cell density and growth patterns) and benign features (such as sclerosing adenosis). Additionally, quantitative micro-elastography (QME) images presented alongside OCT and attenuation images show that these techniques provide complementary contrast, suggesting that multimodal imaging could increase tissue identification accuracy and potentially improve tumor margin assessment.

Handheld volumetric manual compression-based quantitative microelastography.

Compression optical coherence elastography (OCE) typically requires a mechanical actuator to impart a controlled uniform strain to the sample. However, for handheld scanning, this adds complexity to the design of the probe and the actuator stroke limits the amount of strain that can be applied. In this work, we present a new volumetric imaging approach that utilizes bidirectional manual compression via the natural motion of the user's hand to induce strain to the sample, realizing compact, actuator-free, handheld compression OCE. In this way, we are able to demonstrate rapid acquisition of three-dimensional quantitative microelastography (QME) datasets of a tissue volume (6 × 6 × 1 mm3 ) in 3.4 seconds. We characterize the elasticity sensitivity of this freehand manual compression approach using a homogeneous silicone phantom and demonstrate comparable performance to a benchtop mounted, actuator-based approach. In addition, we demonstrate handheld volumetric manual compression-based QME on a tissue-mimicking phantom with an embedded stiff inclusion and on freshly excised human breast specimens from both mastectomy and wide local excision (WLE) surgeries. Tissue results are coregistered with postoperative histology, verifying the capability of our approach to measure the elasticity of tissue and to distinguish stiff tumor from surrounding soft benign tissue.

Diagnostic Accuracy of Quantitative Micro-Elastography for Margin Assessment in Breast-Conserving Surgery.

Inadequate margins in breast-conserving surgery (BCS) are associated with an increased likelihood of local recurrence of breast cancer. Currently, approximately 20% of BCS patients require repeat surgery due to inadequate margins at the initial operation. Implementation of an accurate, intraoperative margin assessment tool may reduce this re-excision rate. This study determined, for the first time, the diagnostic accuracy of quantitative micro-elastography (QME), an optical coherence tomography (OCT)-based elastography technique that produces images of tissue microscale elasticity, for detecting tumor within 1 mm of the margins of BCS specimens. Simultaneous OCT and QME were performed on the margins of intact, freshly excised specimens from 83 patients undergoing BCS and on dissected specimens from 7 patients undergoing mastectomy. The resulting three-dimensional images (45 × 45 × 1 mm) were coregistered with postoperative histology to determine tissue types present in each scan. Data from 12 BCS patients and the 7 mastectomy patients served to build a set of images for reader training. One hundred and fifty-four subimages (10 × 10 × 1 mm) from the remaining 71 BCS patients were included in a blinded reader study, which resulted in 69.0% sensitivity and 79.0% specificity using OCT images, versus 92.9% sensitivity and 96.4% specificity using elasticity images. The quantitative nature of QME also facilitated development of an automated reader, which resulted in 100.0% sensitivity and 97.7% specificity. These results demonstrate high accuracy of QME for detecting tumor within 1 mm of the margin and the potential for this technique to improve outcomes in BCS. SIGNIFICANCE: An optical imaging technology probes breast tissue elasticity to provide accurate assessment of tumor margin involvement in breast-conserving surgery.

Analysis of spatial resolution in phase-sensitive compression optical coherence elastography.

Optical coherence elastography (OCE) is emerging as a method to image the mechanical properties of tissue on the microscale. However, the spatial resolution, a main advantage of OCE, has not been investigated and is not trivial to evaluate. To address this, we present a framework to analyze resolution in phase-sensitive compression OCE that incorporates the three main determinants of resolution: mechanical deformation of the sample, detection of this deformation using optical coherence tomography (OCT), and signal processing to estimate local axial strain. We demonstrate for the first time, through close correspondence between experiment and simulation of structured phantoms, that resolution in compression OCE is both spatially varying and sample dependent, which we link to the discrepancies between the model of elasticity and the mechanical deformation of the sample. We demonstrate that resolution is dependent on factors such as feature size and mechanical contrast. We believe that the analysis of image formation provided by our framework can expedite the development of compression OCE.

Finger-mounted quantitative micro-elastography.

We present a finger-mounted quantitative micro-elastography (QME) probe, capable of measuring the elasticity of biological tissue in a format that avails of the dexterity of the human finger. Finger-mounted QME represents the first demonstration of a wearable elastography probe. The approach realizes optical coherence tomography-based elastography by focusing the optical beam into the sample via a single-mode fiber that is fused to a length of graded-index fiber. The fiber is rigidly affixed to a 3D-printed thimble that is mounted on the finger. Analogous to manual palpation, the probe compresses the tissue through the force exerted by the finger. The resulting deformation is measured using optical coherence tomography. Elasticity is estimated as the ratio of local stress at the sample surface, measured using a compliant layer, to the local strain in the sample. We describe the probe fabrication method and the signal processing developed to achieve accurate elasticity measurements in the presence of motion artifact. We demonstrate the probe's performance in motion-mode scans performed on homogeneous, bi-layer and inclusion phantoms and its ability to measure a thermally-induced increase in elasticity in ex vivo muscle tissue. In addition, we demonstrate the ability to acquire 2D images with the finger-mounted probe where lateral scanning is achieved by swiping the probe across the sample surface.

Handheld probe for quantitative micro-elastography.

Optical coherence elastography (OCE) has been proposed for a range of clinical applications. However, the majority of these studies have been performed using bulky, lab-based imaging systems. A compact, handheld imaging probe would accelerate clinical translation, however, to date, this had been inhibited by the slow scan rates of compact devices and the motion artifact induced by the user's hand. In this paper, we present a proof-of-concept, handheld quantitative micro-elastography (QME) probe capable of scanning a 6 × 6 × 1 mm volume of tissue in 3.4 seconds. This handheld probe is enabled by a novel QME acquisition protocol that incorporates a custom bidirectional scan pattern driving a microelectromechanical system (MEMS) scanner, synchronized with the sample deformation induced by an annular PZT actuator. The custom scan pattern reduces the total acquisition time and the time difference between B-scans used to generate displacement maps, minimizing the impact of motion artifact. We test the feasibility of the handheld QME probe on a tissue-mimicking silicone phantom, demonstrating comparable image quality to a bench-mounted setup. In addition, we present the first handheld QME scans performed on human breast tissue specimens. For each specimen, quantitative micro-elastograms are co-registered with, and validated by, histology, demonstrating the ability to distinguish stiff cancerous tissue from surrounding soft benign tissue.

Clinical feasibility of optical coherence micro-elastography for imaging tumor margins in breast-conserving surgery.

It has been demonstrated that optical coherence micro-elastography (OCME) provides additional contrast of tumor compared to optical coherence tomography (OCT) alone. Previous studies, however, have predominantly been performed on mastectomy specimens. Such specimens typically differ substantially in composition and geometry from the more clinically relevant wide-local excision (WLE) specimens excised during breast-conserving surgery. As a result, it remains unclear if the mechanical contrast observed is maintained in WLE specimens. In this manuscript, we begin to address this issue by performing a feasibility study of OCME on 17 freshly excised, intact WLE specimens. In addition, we present two developments required to sustain the progression of OCME towards intraoperative deployment. First, to enable the rapid visualization of en face images required for intraoperative assessment, we describe an automated segmentation algorithm to fuse en face micro-elastograms with OCT images to provide dual contrast images. Secondly, to validate contrast in micro-elastograms, we present a method that enables co-registration of en face images with histology of WLE specimens, sectioned in the orthogonal plane, without any modification to the standard clinical workflow. We present a summary of the observations across the 17 specimens imaged in addition to representative micro-elastograms and OCT images demonstrating contrast in a number of tumor margins, including those involved by invasive ductal carcinoma, mucinous carcinoma, and solid-papillary carcinoma. The results presented here demonstrate the potential of OCME for imaging tumor margins.

Wide-field quantitative micro-elastography of human breast tissue.

Currently, 20-30% of patients undergoing breast-conserving surgery require a second surgery due to insufficient surgical margins in the initial procedure. We have developed a wide-field quantitative micro-elastography system for the assessment of tumor margins. In this technique, we map tissue elasticity over a field-of-view of ~46 × 46 mm. We performed wide-field quantitative micro-elastography on thirteen specimens of freshly excised tissue acquired from patients undergoing a mastectomy. We present wide-field optical coherence tomography (OCT) images, qualitative (strain) micro-elastograms and quantitative (elasticity) micro-elastograms, acquired in 10 minutes. We demonstrate that wide-field quantitative micro-elastography can extend the range of tumors visible using OCT-based elastography by providing contrast not present in either OCT or qualitative micro-elastography and, in addition, can reduce imaging artifacts caused by a lack of contact between tissue and the imaging window. Also, we describe how the combined evaluation of OCT, qualitative micro-elastograms and quantitative micro-elastograms can improve the visualization of tumor.

Simplifying the assessment of human breast cancer by mapping a micro-scale heterogeneity index in optical coherence elastography.

Surgical treatment of breast cancer aims to identify and remove all malignant tissue. Intraoperative assessment of tumor margins is, however, not exact; thus, re-excision is frequently needed, or excess normal tissue is removed. Imaging methods applicable intraoperatively could help to reduce re-excision rates whilst minimizing removal of excess healthy tissue. Optical coherence elastography (OCE) has been proposed for use in breast-conserving surgery; however, intraoperative interpretation of complex OCE images may prove challenging. Observations of breast cancer on multiple length scales, by OCE, ultrasound elastography, and atomic force microscopy, have shown an increase in the mechanical heterogeneity of malignant breast tumors compared to normal breast tissue. In this study, a micro-scale mechanical heterogeneity index is introduced and used to form heterogeneity maps from OCE scans of 10 ex vivo human breast tissue samples. Through comparison of OCE, optical coherence tomography images, and corresponding histology, malignant tissue is shown to possess a higher heterogeneity index than benign tissue. The heterogeneity map simplifies the contrast between tumor and normal stroma in breast tissue, facilitating the rapid identification of possible areas of malignancy, which is an important step towards intraoperative margin assessment using OCE.

In vivo volumetric quantitative micro-elastography of human skin.

In this paper, we demonstrate in vivo volumetric quantitative micro-elastography of human skin. Elasticity is estimated at each point in the captured volume by combining local axial strain measured in the skin with local axial stress estimated at the skin surface. This is achieved by utilizing phase-sensitive detection to measure axial displacements resulting from compressive loading of the skin and an overlying, compliant, transparent layer with known stress/strain behavior. We use an imaging probe head that provides optical coherence tomography imaging and compression from the same direction. We demonstrate our technique on a tissue phantom containing a rigid inclusion, and present in vivo elastograms acquired from locations on the hand, wrist, forearm and leg of human volunteers.

Investigation of optical attenuation imaging using optical coherence tomography for monitoring of scars undergoing fractional laser treatment.

We demonstrate the use of the near-infrared attenuation coefficient, measured using optical coherence tomography (OCT), in longitudinal assessment of hypertrophic burn scars undergoing fractional laser treatment. The measurement method incorporates blood vessel detection by speckle decorrelation and masking, and a robust regression estimator to produce 2D en face parametric images of the attenuation coefficient of the dermis. Through reliable co-location of the field of view across pre- and post-treatment imaging sessions, the study was able to quantify changes in the attenuation coefficient of the dermis over a period of ∼20 weeks in seven patients. Minimal variation was observed in the mean attenuation coefficient of normal skin and control (untreated) mature scars, as expected. However, a significant decrease (13 ± 5%, mean ± standard deviation) was observed in the treated mature scars, resulting in a greater distinction from normal skin in response to localized damage from the laser treatment. By contrast, we observed an increase in the mean attenuation coefficient of treated (31 ± 27%) and control (27 ± 20%) immature scars, with numerical values incrementally approaching normal skin as the healing progressed. This pilot study supports conducting a more extensive investigation of OCT attenuation imaging for quantitative longitudinal monitoring of scars. En face 2D OCT attenuation coefficient map of a treated immature scar derived from the pre-treatment (top) and the post-treatment (bottom) scans. (Vasculature (black) is masked out.) The scale bars are 0.5 mm.