Jeffrey A. Mulligan

Emerging Approaches for High-Resolution Imaging of Tissue Biomechanics With Optical Coherence Elastography

Optical coherence elastography (OCE) is an emerging method to image the mechanical properties of tissue in 3D, with spatial resolution approaching the micrometer-scale. By offering the ability to connect between the cellular and bulk tissue levels, OCE has the potential to enable biomechanics-based clinical diagnostics and in vivo biomechanics investigations over a new spatial scale. We review the emerging approaches in OCE, emphasizing commonalities with ultrasound elastography and a fundamental connection to the emerging field of Mechanobiology. The concept of a forward and inverse problem is applied to image formation in OCE, and more broadly, to describe overall efforts in the field. We also discuss the potential impact that OCE holds for clinical and biological applications, and highlight future directions for the field, including opportunities for cross-modality and interdisciplinary interactions.

Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy

Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research.

GPU-based computational adaptive optics for volumetric optical coherence microscopy

Optical coherence tomography (OCT) is a non-invasive imaging technique that measures reflectance from within biological tissues. Current higher-NA optical coherence microscopy (OCM) technologies with near cellular resolution have limitations on volumetric imaging capabilities due to the trade-offs between resolution vs. depth-of-field and sensitivity to aberrations. Such trade-offs can be addressed using computational adaptive optics (CAO), which corrects aberration computationally for all depths based on the complex optical field measured by OCT. However, due to the large size of datasets plus the computational complexity of CAO and OCT algorithms, it is a challenge to achieve high-resolution 3D-OCM reconstructions at speeds suitable for clinical and research OCM imaging. In recent years, real-time OCT reconstruction incorporating both dispersion and defocus correction has been achieved through parallel computing on graphics processing units (GPUs). We add to these methods by implementing depth-dependent aberration correction for volumetric OCM using plane-by-plane phase deconvolution. Following both defocus and aberration correction, our reconstruction algorithm achieved depth-independent transverse resolution of 2.8 um, equal to the diffraction-limited focal plane resolution. We have translated the CAO algorithm to a CUDA code implementation and tested the speed of the software in real-time using two GPUs - NVIDIA Quadro K600 and Geforce TITAN Z. For a data volume containing 4096×256×256 voxels, our system’s processing speed can keep up with the 60 kHz acquisition rate of the line-scan camera, and takes 1.09 seconds to simultaneously update the CAO correction for 3 en face planes at user-selectable depths.

Quantitative reconstruction of time-varying 3D cell forces with traction force optical coherence microscopy

Cellular traction forces (CTFs) play an integral role in both physiological processes and disease, and are a topic of interest in mechanobiology. Traction force microscopy (TFM) is a family of methods used to quantify CTFs in a variety of settings. State-of-the-art 3D TFM methods typically rely on confocal fluorescence microscopy, which can impose limitations on acquisition speed, volumetric coverage, and temporal sampling or coverage. In this report, we present the first quantitative implementation of a new TFM technique: traction force optical coherence microscopy (TF-OCM). TF-OCM leverages the capabilities of optical coherence microscopy and computational adaptive optics (CAO) to enable the quantitative reconstruction of 3D CTFs in scattering media with minute-scale temporal resolution. We applied TF-OCM to quantify CTFs exerted by isolated NIH-3T3 fibroblasts embedded in Matrigel, with five-minute temporal sampling, using images which spanned a 500×500×500 μm3 field-of-view. Due to the reliance of TF-OCM on computational imaging methods, we have provided extensive discussion of the underlying equations, assumptions, and failure modes of these methods. TF-OCM has the potential to advance studies of biomechanical behavior in scattering media, and may be especially well-suited to the study of cell collectives such as spheroids, a prevalent model in mechanobiology research.

Traction Force Microscopy for Noninvasive Imaging of Cell Forces

The forces exerted by cells on their surroundings play an integral role in both physiological processes and disease progression. Traction force microscopy is a noninvasive technique that enables the in vitro imaging and quantification of cell forces. Utilizing expertise from a variety of disciplines, recent developments in traction force microscopy are enhancing the study of cell forces in physiologically relevant model systems, and hold promise for further advancing knowledge in mechanobiology. In this chapter, we discuss the methods, capabilities, and limitations of modern approaches for traction force microscopy, and highlight ongoing efforts and challenges underlying future innovations.

Volumetric optical coherence microscopy enabled by aberrated optics (Conference Presentation)

Optical coherence microscopy (OCM) is an interferometric imaging technique that enables high resolution, non-invasive imaging of 3D cell cultures and biological tissues. Volumetric imaging with OCM suffers a trade-off between high transverse resolution and poor depth-of-field resulting from defocus, optical aberrations, and reduced signal collection away from the focal plane. While defocus and aberrations can be compensated with computational methods such as interferometric synthetic aperture microscopy (ISAM) or computational adaptive optics (CAO), reduced signal collection must be physically addressed through optical hardware. Axial scanning of the focus is one approach, but comes at the cost of longer acquisition times, larger datasets, and greater image reconstruction times. Given the capabilities of CAO to compensate for general phase aberrations, we present an alternative method to address the signal collection problem without axial scanning by using intentionally aberrated optical hardware. We demonstrate the use of an astigmatic spectral domain (SD-)OCM imaging system to enable single-acquisition volumetric OCM in 3D cell culture over an extended depth range, compared to a non-aberrated SD-OCM system. The transverse resolution of the non-aberrated and astigmatic imaging systems after application of CAO were 2 um and 2.2 um, respectively. The depth-range of effective signal collection about the nominal focal plane was increased from 100 um in the non-aberrated system to over 300 um in the astigmatic system, extending the range over which useful data may be acquired in a single OCM dataset. We anticipate that this method will enable high-throughput cellular-resolution imaging of dynamic biological systems over extended volumes.

Measurement of time-varying displacement fields in cell culture for traction force optical coherence microscopy (Conference Presentation)

Mechanobiology is an emerging field which seeks to link mechanical forces and properties to the behaviors of cells and tissues in cancer, stem cell growth, and other processes. Traction force microscopy (TFM) is an imaging technique that enables the study of traction forces exerted by cells on their environment to migrate as well as sense and manipulate their surroundings. To date, TFM research has been performed using incoherent imaging modalities and, until recently, has been largely confined to the study of cell-induced tractions within two-dimensions using highly artificial and controlled environments. As the field of mechanobiology advances, and demand grows for research in physiologically relevant 3D culture and in vivo models, TFM will require imaging modalities that support such settings. Optical coherence microscopy (OCM) is an interferometric imaging modality which enables 3D cellular resolution imaging in highly scattering environments. Moreover, optical coherence elastography (OCE) enables the measurement of tissue mechanical properties. OCE relies on the principle of measuring material deformations in response to artificially applied stress. By extension, similar techniques can enable the measurement of cell-induced deformations, imaged with OCM. We propose traction force optical coherence microscopy (TF-OCM) as a natural extension and partner to existing OCM and OCE methods. We report the first use of OCM data and digital image correlation to track temporally varying displacement fields exhibited within a 3D culture setting. These results mark the first steps toward the realization of TF-OCM in 2D and 3D settings, bolstering OCM as a platform for advancing research in mechanobiology.

Computational adaptive optics for high-throughput volumetric optical coherence microscopy

High-throughput optical imaging is playing an increasingly important role in biological imaging. The technique enables biological dynamics to be studied at a variety of different spatiotemporal scales.1–3 One area that is poised to benefit from this capability is the study of collective or emergent behavior in embryonic development, tissue regeneration, and cancer.4, 5 Existing modalities for high-speed volumetric imaging at cellular or sub-cellular resolution are typically based on the detection of fluorescence signals. Consequently, they are subject to photobleaching and phototoxicity constraints and, as a result of this, have limited scope in settings that preclude the use of exogenous contrast agents (e.g., many clinical settings). High-speed methods such as optical coherence tomography (OCT) could help to provide label-free imaging of biological dynamics, thereby filling this gap in biological imaging. Recent advances in ultrahigh-speed OCT have enabled the acquisition of volumetric datasets at video rates (i.e., one volume in 25ms).6 However, combining this high-throughput acquisition with cellular-resolution optical coherence microscopy (OCM) presents significant challenges. The main factor limiting the volumetric acquisition rate of OCM is the rapid degradation of resolution and signal strength at increased distance from the point of optical focus. There are a number of hardware approaches to volumetric cellular-resolution OCM. These include the acquisition of multiple OCM datasets, each with a different focus depth, and subsequent synthesis of a single ‘in-focus’ volume.7–9 The illumination and collection beam can also be engineered to provide an extended focal region.10 Of these approaches, focus-scanning methods offer the highest Figure 1. Volumetric optical coherence microscopy (OCM) and CAOOCM (computational adaptive optics-OCM) reconstructions of a grape sample, imaged with an astigmatic system. (a) An image of the volume obtained via OCM and (b) the corresponding CAO-OCM reconstruction. Volume renderings show a depth range spanning from 220 to 1130 m below the sample surface. These reconstructions and volume renderings were performed offline. (c–e) Real-time en face OCM planes from three depths (380, 730, and 1030 m, respectively), obtained using a graphics processing unit. (f–h) The corresponding CAO-OCM en face planes, showing simultaneous real-time correction of defocus and astigmatism. Scale bars: 100 m.