Logarithmic intensity and speckle-based motion contrast methods for human retinal vasculature visualization using swept source optical coherence tomography

We formulate a theory to show that the statistics of OCT signal amplitude and intensity are highly dependent on the sample reflectivity strength, motion, and noise power. Our theoretical and experimental results depict the lack of speckle amplitude and intensity contrasts to differentiate regions of motion from static areas. Two logarithmic intensity-based contrasts, logarithmic intensity variance (LOGIV) and differential logarithmic intensity variance (DLOGIV), are proposed for serving as surrogate markers for motion with enhanced sensitivity. Our findings demonstrate a good agreement between the theoretical and experimental results for logarithmic intensity-based contrasts. Logarithmic intensity-based motion and speckle-based contrast methods are validated and compared for in vivo human retinal vasculature visualization using high-speed swept-source optical coherence tomography (SS-OCT) at 1060 nm. The vasculature was identified as regions of motion by creating LOGIV and DLOGIV tomograms: multiple B-scans were collected of individual slices through the retina and the variance of logarithmic intensities and differences of logarithmic intensities were calculated. Both methods captured the small vessels and the meshwork of capillaries associated with the inner retina in en face images over 4 mm^2 in a normal subject

Polarimetry noise in fiber-based optical coherence tomography instrumentation

High noise levels in fiber-based polarization-sensitive optical coherence tomography (PS-OCT) have broadly limited its clinical utility. In this study we investigate contribution of polarization mode dispersion (PMD) to the polarimetry noise. We develop numerical models of the PS-OCT system including PMD and validate these models with empirical data. Using these models, we provide a framework for predicting noise levels, for processing signals to reduce noise, and for designing an optimized system

C/EBPalpha: critical at the origin of leukemic transformation

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by clonal expansion of myeloid progenitor cells. A major mechanistic theme in AML biology is the extensive collaboration among fusion oncoproteins, transcription factors, and chromatin regulators to initiate and sustain a transformed cellular state. A new study in this issue describes how the C/EBPalpha transcription factor is crucial for the initiation of AML induced by MLL fusion oncoproteins, but is entirely dispensable for the maintenance of established disease. These observations provide a unique glimpse into the pioneer round of regulatory events that are critical at the origin of AML formation. Furthermore, this study implies the existence of oncogene-induced positive feedback loops capable of bypassing the continuous need for certain regulators to propagate disease

In vivo volumetric imaging of human retinal circulation with phase-variance optical coherence tomography

We present in vivo volumetric images of human retinal micro-circulation using Fourier-domain optical coherence tomography (Fd-OCT) with the phase-variance based motion contrast method. Currently fundus fluorescein angiography (FA) is the standard technique in clinical settings for visualizing blood circulation of the retina. High contrast imaging of retinal vasculature is achieved by injection of a fluorescein dye into the systemic circulation. We previously reported phase-variance optical coherence tomography (pvOCT) as an alternative and non-invasive technique to image human retinal capillaries. In contrast to FA, pvOCT allows not only noninvasive visualization of a two-dimensional retinal perfusion map but also volumetric morphology of retinal microvasculature with high sensitivity. In this paper we report high-speed acquisition at 125 kHz A-scans with pvOCT to reduce motion artifacts and increase the scanning area when compared with previous reports. Two scanning schemes with different sampling densities and scanning areas are evaluated to find optimal parameters for high acquisition speed in vivo imaging. In order to evaluate this technique, we compare pvOCT capillary imaging at 3x3 mm^2 and 1.5x1.5 mm^2 with fundus FA for a normal human subject. Additionally, a volumetric view of retinal capillaries and a stitched image acquired with ten 3x3 mm^2 pvOCT sub-volumes are presented. Visualization of retinal vasculature with pvOCT has potential for diagnosis of retinal vascular diseases

Optical-domain subsampling for data efficient depth ranging in Fourier-domain optical coherence tomography

Recent advances in optical coherence tomography (OCT) have led to higher-speed sources that support imaging over longer depth ranges. Limitations in the bandwidth of state-of-the-art acquisition electronics, however, prevent adoption of these advances into the clinical applications. Here, we introduce optical-domain subsampling as a method for imaging at high-speeds and over extended depth ranges but with a lower acquisition bandwidth than that required using conventional approaches. Optically subsampled laser sources utilize a discrete set of wavelengths to alias fringe signals along an extended depth range into a bandwidth limited frequency window. By detecting the complex fringe signals and under the assumption of a depth-constrained signal, optical-domain subsampling enables recovery of the depth-resolved scattering signal without overlapping artifacts from this bandwidth-limited window. We highlight key principles behind optical-domain subsampled imaging, and demonstrate this principle experimentally using a polygon-filter based swept-source laser that includes an intra-cavity Fabry-Perot (FP) etalon

ACE: A Probabilistic Model for Characterizing Gene-Level Essentiality in CRISPR Screens

High-throughput knockout screens based on CRISPR-Cas9 are widely used to evaluate the essentiality of genes across a range of cell types. Here we introduce a probabilistic modeling framework, Analysis of CRISPR-based Essentiality (ACE), that enables new statistical tests for essentiality based on the raw sequence read counts from such screens. ACE estimates the essentiality of each gene using a flexible likelihood framework that accounts for multiple sources of variation in the CRISPR-Cas9 experimental process. In addition, the method can identify genes that differ in their degree of essentiality across samples using a likelihood ratio test. We show using simulations that ACE is competitive with the best available methods in predicting essentiality, and is especially useful for the identification of differential essentiality. Furthermore, by applying ACE to publicly available CRISPR-screen data, we are able to identify both known and previously overlooked candidates for genotype-specific essentiality, including RNA m 6 -A methyltransferases that exhibit enhanced essentiality in the presence of inactivating TP53 mutations. In summary, ACE provides improved quantification of essentiality specific to cancer subtypes, and a robust probabilistic framework for identifying genes responsive to therapeutic targeting

Photometric stereo endoscopy

While color video endoscopy has enabled wide-field examination of the gastrointestinal tract, it often misses or incorrectly classifies lesions. Many of these missed lesions exhibit characteristic three-dimensional surface topographies. An endoscopic system that adds topographical measurements to conventional color imagery could therefore increase lesion detection and improve classification accuracy. We introduce photometric stereo endoscopy (PSE), a technique which allows high spatial frequency components of surface topography to be acquired simultaneously with conventional two-dimensional color imagery. We implement this technique in an endoscopic form factor and demonstrate that it can acquire the topography of small features with complex geometries and heterogeneous optical properties. PSE imaging of ex vivo human gastrointestinal tissue shows that surface topography measurements enable differentiation of abnormal shapes from surrounding normal tissue. Together, these results confirm that the topographical measurements can be obtained with relatively simple hardware in an endoscopic form factor, and suggest the potential of PSE to improve lesion detection and classification in gastrointestinal imaging.This work has been financially supported by the Comunidad de Madrid through the Madrid-MIT M+Visión Consortium

Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis

Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development

Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia

Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program

ACE: a probabilistic model for characterizing gene-level essentiality in CRISPR screens.

High-throughput CRISPR-Cas9 knockout screens are widely used to evaluate gene essentiality in cancer research. Here we introduce a probabilistic modeling framework, Analysis of CRISPR-based Essentiality (ACE), that accounts for multiple sources of variation in CRISPR-Cas9 screens and enables new statistical tests for essentiality. We show using simulations that ACE is effective at predicting both absolute and differential essentiality. When applied to publicly available data, ACE identifies known and novel candidates for genotype-specific essentiality, including RNA m6-A methyltransferases that exhibit enhanced essentiality in the presence of inactivating TP53 mutations. ACE provides a robust framework for identifying genes responsive to subtype-specific therapeutic targeting