<p>Recent advances in fluorescence  microscopy enable deeper cellular imaging in living tissues with near-infrared excitation light. </p> <p>High quality fluorescence microscopy images provide useful information for analyzing biological structures and diagnosing diseases.</p> <p>Nuclei detection and segmentation are two fundamental steps for quantitative analysis of microscopy images.</p> <p>However, existing machine learning-based approaches are hampered by three main challenges: (1) Hand annotated ground truth is difficult to obtain especially for 3D volumes, (2) Most of the object detection methods work only on 2D images and are difficult to extend to 3D volumes, (3) Segmentation-based approaches typically cannot distinguish different object instances without proper post-processing steps.</p> <p>In this thesis, we propose various new methods for microscopy image analysis including nuclei synthesis, detection, and segmentation. </p> <p>Due to the limitation of manually annotated ground truth masks, we first describe how we generate 2D/3D synthetic microscopy images using SpCycleGAN and use them as a data augmentation technique for our detection and segmentation networks.</p> <p>For nuclei detection, we describe our RCNN-SliceNet for nuclei counting and centroid detection using slice-and-cluster strategy. </p> <p>Then we introduce our 3D CentroidNet for nuclei centroid estimation using vector flow voting mechanism which does not require any post-processing steps.</p> <p>For nuclei segmentation, we first describe our EMR-CNN for nuclei instance segmentation using ensemble learning and slice fusion strategy.</p> <p>Then we present the 3D Nuclei Instance Segmentation Network (NISNet3D) for nuclei instance segmentation using gradient vector field array.</p> <p>Extensive experiments have been conducted on a variety of challenging microscopy volumes to demonstrate that our approach can accurately detect and segment the cell nuclei and outperforms other compared methods.</p> <p>Finally, we describe the Distributed and Networked Analysis of Volumetric Image Data (DINAVID) system we developed for biologists to remotely analyze large microscopy volumes using machine learning. </p>

<div>Fluorescence microscopy has emerged as a powerful tool for studying cell biology because it enables the acquisition of 3D image volumes deeper into tissue and the imaging of complex subcellular structures. Fluorescence microscopy images are frequently distorted by motion resulting from animal respiration and heartbeat which complicates the quantitative analysis of biological structures needed to characterize the structure and constituency of tissue volumes. This thesis describes a two pronged approach to quantitative analysis consisting of non-rigid registration and deep convolutional neural network segmentation. The proposed image registration method is capable of correcting motion artifacts in three dimensional fluorescence microscopy images collected over time. In particular, our method uses 3D B-Spline based nonrigid registration using a coarse-to-fine strategy to register stacks of images collected at different time intervals and 4D rigid registration to register 3D volumes over time. The results show that the proposed method has the ability of correcting global motion artifacts of sample tissues in four dimensional space, thereby revealing the motility of individual cells in the tissue.</div><div><br></div><div>We describe in thesis nuclei segmentation methods using deep convolutional neural networks, data augmentation to generate training images of different shapes and contrasts, a refinement process combining segmentation results of horizontal, frontal, and sagittal planes in a volume, and a watershed technique to enumerate the nuclei. Our results indicate that compared to 3D ground truth data, our method can successfully segment and count 3D nuclei. Furthermore, a microscopy image synthesis method based on spatially constrained cycle-consistent adversarial networks is used to efficiently generate training data. A 3D modified U-Net network is trained with a combination of Dice loss and binary cross entropy metrics to achieve accurate nuclei segmentation. A multi-task U-Net is utilized to resolve overlapping nuclei. This method was found to achieve high accuracy object-based and voxel-based evaluations.</div>