Optimization of a flexible fiber-optic probe for epi-mode quantitative phase imaging.

Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is <20nm for a single qOBM acquisition (at 10Hz) and a lower limit of ∼3 nm via multi-frame averaging. Finally, to demonstrate the utility of the optimized probe, we image (1) thick, fixed rat brain samples from a 9L gliosarcoma tumor model and (2) freshly excised human brain tissues from neurosurgery. Acquired qOBM images using the flexible fiber-optic probe are in excellent agreement with those from a free-space qOBM system (both in-situ), as well as with gold-standard histopathology slices (after tissue processing).

Noninvasive white blood cell quantification in umbilical cord blood collection bags with quantitative oblique back-illumination microscopy.

BACKGROUND: Umbilical cord blood has become an important source of hematopoietic stem and progenitor cells for therapeutic applications. However, cord blood banking (CBB) grapples with issues related to economic viability, partially due to high discard rates of cord blood units (CBUs) that lack sufficient total nucleated cells for storage or therapeutic use. Currently, there are no methods available to assess the likelihood of CBUs meeting storage criteria noninvasively at the collection site, which would improve CBB efficiency and economic viability. MATERIALS AND METHODS: To overcome this limitation, we apply a novel label-free optical imaging method, called quantitative oblique back-illumination microscopy (qOBM), which yields tomographic phase and absorption contrast to image blood inside collection bags. An automated segmentation algorithm was developed to count white blood cells and red blood cells (RBCs) and assess hematocrit. Fifteen CBUs were measured. RESULTS: qOBM clearly differentiates between RBCs and nucleated cells. The cell-counting analysis shows an average error of 13% compared to hematology analysis, with a near-perfect, one-to-one relationship (slope = 0.94) and strong correlation coefficient (r = 0.86). Preliminary results to assess hematocrit also show excellent agreement with expected values. Acquisition times to image a statistically significant number of cells per CBU were approximately 1 minute. CONCLUSION: qOBM exhibits robust performance for quantifying blood inside collection bags. Because the approach is automated and fast, it can potentially quantify CBUs within minutes of collection, without breaching the CBUs' sterile environment. qOBM can reduce costs in CBB by avoiding processing expenses of CBUs that ultimately do not meet storage criteria.

Coherently broadened, high-repetition-rate laser for stimulated Raman scattering-spectroscopic optical coherence tomography.

We present a novel light source specifically tailored for stimulated Raman scattering-spectroscopic optical coherence tomography (SRS-SOCT), which is, to the best of our knowledge, a novel molecular imaging method that combines the molecular sensitivity of SRS with the spatial and spectral multiplexing capabilities of SOCT. The novel laser consists of an 8 W, 450 fs Yb:KGW oscillator, with a repetition rate of 40 MHz, which delivers the Stokes beam for SRS-SOCT and also pumps and amplifies an optical parametric oscillator (OPO). The output of the amplified OPO is then frequency doubled and coherently broadened using a custom-made tapered fiber that generates bandwidth pulses >40 nm, compressible to <50 fs, with the average power over 150 mW, near the shot-noise limit above 250 kHz. The broadened and compressed pulse simultaneously serves as the pump beam and SOCT light source for SRS-SOCT. This light source is assessed for SRS-SOCT, and its implications for other imaging methods are discussed.

Cellular effects of acute exposure to high peak power microwave systems: Morphology and toxicology.

Electric fields produced by advanced pulsed microwave transmitter technology now readily exceed the Institute of Electrical and Electronic Engineers (IEEE) C.95.1 peak E-field limit of 100 kV/m, highlighting a need for scientific validation of such a specific limit. Toward this goal, we exposed Jurkat Clone E-6 human lymphocyte preparations to 20 high peak power microwave (HPPM) pulses (120 ns duration) with a mean peak amplitude of 2.3 MV/m and standard deviation of 0.1 with the electric field at cells predicted to range from 0.46 to 2.7 MV/m, well in excess of current standard limit. We observed that membrane integrity and cell morphology remained unchanged 4 h after exposure and cell survival 24 h after exposure was not statistically different from sham exposure or control samples. Using flow cytometry to analyze membrane disruption and morphological changes per exposed cell, no changes were observed in HPPM-exposed samples. Current IEEE C95.1-2005 standards for pulsed radiofrequency exposure limits peak electric field to 100 kV/m for pulses shorter than 100 ms [IEEE (1995) PC95.1-Standard for Safety Levels with Respect to Human Exposure to Electric, Magnetic and Electromagnetic Fields, 0 Hz to 300 GHz, Institute of Electrical and Electronic Engineers: Piscataway, NJ, USA]. This may impose large exclusion zones that limit HPPM technology use. In this study, we offer evidence that maximum permissible exposure of 100 kV/m for peak electric field may be unnecessarily restrictive for HPPM devices. Bioelectromagnetics. 37:141-151, 2016. © 2016 Wiley Periodicals, Inc.

Partially coherent broadband 3D optical transfer functions with arbitrary temporal and angular power spectra.

Optical diffraction tomography is a powerful technique to produce 3D volumetric images of biological samples using contrast produced by variations in the index of refraction in an unlabeled specimen. While this is typically performed with coherent illumination from a variety of angles, interest has grown in partially coherent methods due to the simplicity of the illumination and the computation-free axial sectioning provided by the coherence window of the source. However, such methods rely on the symmetry or discretization of a source to facilitate quantitative analysis and are unable to efficiently handle arbitrary illumination that may vary asymmetrically in angle and continuously in the spectrum, such as diffusely scattered or thermal sources. A general broadband theory may expand the scope of illumination methods available for quantitative analysis, as partially coherent sources are commonly available and may benefit from the effects of spatial and temporal incoherence. In this work, we investigate partially coherent tomographic phase microscopy from arbitrary sources regardless of angular distribution and spectrum by unifying the effects of spatial and temporal coherence into a single formulation. This approach further yields a method for efficient computation of the overall systems' optical transfer function, which scales with O(n 3), down from O(mn 4) for existing convolutional methods, where n 3 is the number of spatial voxels in 3D space and m is the number of discrete wavelengths in the illumination spectrum. This work has important implications for enabling partially coherent 3D quantitative phase microscopy and refractive index tomography in virtually any transmission or epi-illumination microscope.

Quantitative 3D refractive index tomography of opaque samples in epi-mode.

Three-dimensional (3D) refractive index (RI) tomography has recently become an exciting new tool for biological studies. However, its limitation to (1) thin samples resulting from a need of transmissive illumination and (2) small fields of view (typically ~50 µm × 50 µm) has hindered its utility in broader biomedical applications. In this work, we demonstrate 3D RI tomography with a large field of view in opaque, arbitrarily thick scattering samples (unsuitable for imaging with conventional transmissive tomographic techniques) with a penetration depth of ca. one mean free scattering path length (~100 µm in tissue) using a simple, low-cost microscope system with epi-illumination. This approach leverages a solution to the inverse scattering problem via the general non-paraxial 3D optical transfer function of our quantitative oblique back-illumination microscopy (qOBM) optical system. A theoretical analysis is presented along with simulations and experimental validations using polystyrene beads, and rat and human thick brain tissues. This work has significant implications for the investigation of optically thick, semi-infinite samples in a non-invasive and label-free manner. This unique 3D qOBM approach can extend the utility of 3D RI tomography for translational and clinical medicine.

Towards in-vivo label-free detection of brain tumor margins with epi-illumination tomographic quantitative phase imaging.

Brain tumor surgery involves a delicate balance between maximizing the extent of tumor resection while minimizing damage to healthy brain tissue that is vital for neurological function. However, differentiating between tumor, particularly infiltrative disease, and healthy brain in-vivo remains a significant clinical challenge. Here we demonstrate that quantitative oblique back illumination microscopy (qOBM)-a novel label-free optical imaging technique that achieves tomographic quantitative phase imaging in thick scattering samples-clearly differentiates between healthy brain tissue and tumor, including infiltrative disease. Data from a bulk and infiltrative brain tumor animal model show that qOBM enables quantitative phase imaging of thick fresh brain tissues with remarkable cellular and subcellular detail that closely resembles histopathology using hematoxylin and eosin (H&E) stained fixed tissue sections, the gold standard for cancer detection. Quantitative biophysical features are also extracted from qOBM which yield robust surrogate biomarkers of disease that enable (1) automated tumor and margin detection with high sensitivity and specificity and (2) facile visualization of tumor regions. Finally, we develop a low-cost, flexible, fiber-based handheld qOBM device which brings this technology one step closer to in-vivo clinical use. This work has significant implications for guiding neurosurgery by paving the way for a tool that delivers real-time, label-free, in-vivo brain tumor margin detection.

Epi-mode tomographic quantitative phase imaging in thick scattering samples.

Quantitative phase imaging (QPI) is an important tool in biomedicine that allows for the microscopic investigation of live cells and other thin, transparent samples. Importantly, this technology yields access to the cellular and sub-cellular structure and activity at nanometer scales without labels or dyes. Despite this unparalleled ability, QPI's restriction to relatively thin samples severely hinders its versatility and overall utility in biomedicine. Here we overcome this significant limitation of QPI to enable the same rich level of quantitative detail in thick scattering samples. We achieve this by first illuminating the sample in an epi-mode configuration and using multiple scattering within the sample-a hindrance to conventional transmission imaging used in QPI-as a source of transmissive illumination from within. Second, we quantify phase via deconvolution by modeling the transfer function of the system based on the ensemble average angular distribution of light illuminating the sample at the focal plane. This technique packages the quantitative, real-time sub-cellular imaging capabilities of QPI into a flexible configuration, opening the door for truly non-invasive, label-free, tomographic quantitative phase imaging of unaltered thick, scattering specimens. Images of controlled scattering phantoms, blood in collection bags, cerebral organoids and freshly excised whole mouse brains are presented to validate the approach.

Dual-wavelength oblique back-illumination microscopy for the non-invasive imaging and quantification of blood in collection and storage bags.

There is currently no low-cost method to quantitatively assess the contents of a blood bag without breaching the bag and potentially damaging the sample. Towards this end, we adapt oblique back-illumination microscopy (OBM) to rapidly, inexpensively, and non-invasively screen blood bags for red blood cell (RBC) morphology and white blood cell (WBC) count. OBM has been recently introduced as a tomographic technique that produces high-resolution wide-field images based on phase-gradient and transmission. Here we modify this technique to include illumination at dual wavelengths to facilitate spectral analysis for cell classification. Further, we apply a modified 2D Hilbert transform to recover the phase information from the phase-gradient images for facile cell segmentation. Blood cells are classified as WBCs and RBCs, and counted based on shape, absorption spectrum, and phase profile using an automated algorithm. This work has important implications for the non-invasive assessment of (1) cell viability in storage bags for transfusion applications and (2) suitability of a cord blood collection bag for stem cell therapy applications.