Michael G. Giacomelli

Deformation of stem cell nuclei by nanotopographical cues

Cells sense cues in their surrounding microenvironment. These cues are converted into intracellular signals and transduced to the nucleus in order for the cell to respond and adapt its function. Within the nucleus, structural changes occur that ultimately lead to changes in the gene expression. In this study, we explore the structural changes of the nucleus of human mesenchymal stem cells as an effect of topographical cues. We use a controlled nanotopography to drive shape changes to the cell nucleus, and measure the changes with both fluorescence microscopy and a novel light scattering technique. The nucleus changes shape dramatically in response to the nanotopography, and in a manner dependent on the mechanical properties of the substrate. The kinetics of the nuclear deformation follows an unexpected trajectory. As opposed to a gradual shape change in response to the topography, once the cytoskeleton attains an aligned and elongation morphology on the time scale of several hours, the nucleus changes shape rapidly and intensely.

Label-Free, High-Throughput Measurements of Dynamic Changes in Cell Nuclei Using Angle-Resolved Low Coherence Interferometry

Accurate measurements of nuclear deformation, i.e., structural changes of the nucleus in response to environmental stimuli, are important for signal transduction studies. Traditionally, these measurements require labeling and imaging, and then nuclear measurement using image analysis. This approach is time-consuming, invasive, and unavoidably perturbs cellular systems. Light scattering, an emerging biophotonics technique for probing physical characteristics of living systems, offers a promising alternative. Angle-resolved low-coherence interferometry (a/LCI), a novel light scattering technique, was developed to quantify nuclear morphology for early cancer detection. In this study, a/LCI is used for the first time to noninvasively measure small changes in nuclear morphology in response to environmental stimuli. With this new application, we broaden the potential uses of a/LCI by demonstrating high-throughput measurements and by probing aspherical nuclei. To demonstrate the versatility of this approach, two distinct models relevant to current investigations in cell and tissue engineering research are used. Structural changes in cell nuclei due to subtle environmental stimuli, including substrate topography and osmotic pressure, are profiled rapidly without disrupting the cells or introducing artifacts associated with traditional measurements. Accuracy > or = 3% is obtained for the range of nuclear geometries examined here, with the greatest deviations occurring for the more complex geometries. Given the high-throughput nature of the measurements, this deviation may be acceptable for many biological applications that seek to establish connections between morphology and function.

Application of the T-matrix method to determine the structure of spheroidal cell nuclei with angle-resolved light scattering

We demonstrate an inverse light-scattering analysis procedure based on using the T-matrix method as a light-scattering model. We measure light scattered by in vitro cell monolayers using angle-resolved low-coherence interferometry (a/LCI) and compare the data to predictions of the T-matrix theory. The comparison yields measurements of the equal volume diameter and aspect ratio of the spheroid cell nuclei with accuracy comparable to quantitative image analysis of fixed and stained samples. These improvements represent a significant upgrade for the a/LCI technique, expanding both the range of tissue in which it is applicable and potentially increasing its value as a diagnostic tool.

Application of Mie theory to assess structure of spheroidal scattering in backscattering geometries

Inverse light scattering analysis seeks to associate measured scattering properties with the most probable theoretical scattering distribution. Although Mie theory is a spherical scattering model, it has been used successfully for discerning the geometry of spheroidal scatterers. The goal of this study was an in-depth evaluation of the consequences of analyzing the structure of spheroidal geometries, which are relevant to cell and tissue studies in biology, by employing Mie-theory-based inverse light scattering analysis. As a basis for this study, the scattering from spheroidal geometries was modeled using T-matrix theory and used as test data. In a previous study, we used this technique to investigate the case of spheroidal scatterers aligned with the optical axis. In the present study, we look at a broader scope which includes the effects of aspect ratio, orientation, refractive index, and incident light polarization. Over this wide range of parameters, our results indicate that this method provides a good estimate of spheroidal structure.

Experimental verification of T-matrix-based inverse light scattering analysis for assessing structure of spheroids as models of cell nuclei.

Inverse light scattering analysis (ILSA) seeks to associate measured scattering properties with the most probable theoretical scattering distribution, making it a useful tool for assessing structure in biological materials. The accuracy of ILSA depends on the compatibility of the light scattering geometry with the light scattering model. In this study, we compare the accuracy obtained when analyzing light scattering data from spheroids using a numerical implementation of Mie theory, and the T matrix, a numerical method of solving light scattering from spheroids. Our experimental data are acquired using novel optical phantoms containing spheroidal scatterers and angle-resolved low-coherence interferometry, a depth- and angle-resolved light scattering measurement modality. The results show that Mie theory can accurately assess spheroidal structure despite the geometric incompatibility provided measurements are taken in multiple orientations of the sample relative to the incident polarization and the measured scattering angle. In comparison, analysis using the T-matrix method is highly accurate and more reliable yet requires measurements from only a single orientation.

Size and shape determination of spheroidal scatterers using two-dimensional angle resolved scattering

We demonstrate accurate determination of the size and shape of spherical and spheroidal scatterers through inverse analysis of two-dimensional solid-angle and depth resolved backscattered light intensities. Intensity of scattered light is measured over a wide range of solid angles using a novel scanning fiber optic interferometer from both individual and ensembles of scatterers. T-matrix based inverse analysis of these two-dimensional angular measurements yields completely unique size and aspect ratio determinations with subwavelength precision over a large range of possible scatterer geometries.

Ultrahigh speed en face OCT capsule for endoscopic imaging

Depth resolved and en face OCT visualization in vivo may have important clinical applications in endoscopy. We demonstrate a high speed, two-dimensional (2D) distal scanning capsule with a micromotor for fast rotary scanning and a pneumatic actuator for precision longitudinal scanning. Longitudinal position measurement and image registration were performed by optical tracking of the pneumatic scanner. The 2D scanning device enables high resolution imaging over a small field of view and is suitable for OCT as well as other scanning microscopies. Large field of view imaging for screening or surveillance applications can also be achieved by proximally pulling back or advancing the capsule while scanning the distal high-speed micromotor. Circumferential en face OCT was demonstrated in living swine at 250 Hz frame rate and 1 MHz A-scan rate using a MEMS tunable VCSEL light source at 1300 nm. Cross-sectional and en face OCT views of the upper and lower gastrointestinal tract were generated with precision distal pneumatic longitudinal actuation as well as proximal manual longitudinal actuation. These devices could enable clinical studies either as an adjunct to endoscopy, attached to an endoscope, or as a swallowed tethered capsule for non-endoscopic imaging without sedation. The combination of ultrahigh speed imaging and distal scanning capsule technology could enable both screening and surveillance applications.

Correction of rotational distortion for catheter-based en face OCT and OCT angiography

We demonstrate a computationally efficient method for correcting the nonuniform rotational distortion (NURD) in catheter-based imaging systems to improve endoscopic en face optical coherence tomography (OCT) and OCT angiography. The method performs nonrigid registration using fiducial markers on the catheter to correct rotational speed variations. Algorithm performance is investigated with an ultrahigh-speed endoscopic OCT system and micromotor catheter. Scan nonuniformity is quantitatively characterized, and artifacts from rotational speed variations are significantly reduced. Furthermore, we present endoscopic en face OCT and OCT angiography images of human gastrointestinal tract in vivo to demonstrate the image quality improvement using the correction algorithm.

Ultrahigh speed endoscopic optical coherence tomography for gastroenterology

We describe an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor imaging catheter. The system had a 600 kHz axial scan rate and 8 µm axial resolution in tissue. Imaging was performed with a 3.2 mm diameter imaging catheter at 400 frames per second with a 12 µm spot size. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing upper and lower endoscopy. The use of distally actuated imaging catheters enabled OCT imaging with more flexibility, such as volumetric imaging in the small intestine and the assessment of hiatal hernia using retroflex imaging. The high rotational scanning stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face OCT and cross-sectional imaging, as well as OCT angiography (OCTA) for 3D visualization of subsurface microvasculature. The ability to perform both structural and functional 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies and enhance the sensitivity and specificity of OCT for detecting pathology.

Optical Spectroscopy of Biological Cells

Optical spectroscopy has seen expanding use for the study of biological cells in recent years. An overview of relevant spectroscopic techniques is presented, and applications to biological cells are reviewed.