Identifying spatial imaging biomarkers of glioblastoma multiforme for survival group prediction.

PURPOSE: Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Most GBMs exhibit extensive regional heterogeneity at tissue, cellular, and molecular scales, but the clinical relevance of the observed spatial imaging characteristics remains unknown. We investigated pretreatment magnetic resonance imaging (MRI) scans of GBMs to identify tumor subregions and quantify their image-based spatial characteristics that are associated with survival time. MATERIALS AND METHODS: We quantified tumor subregions (termed habitats) in GBMs, which are hypothesized to capture intratumoral characteristics using multiple MRI sequences. For proof-of-concept, we developed a computational framework that used intratumoral grouping and spatial mapping to identify GBM tumor subregions and yield habitat-based features. Using a feature selector and three classifiers, experimental results from two datasets are reported, including Dataset1 with 32 GBM patients (594 tumor slices) and Dataset2 with 22 GBM patients, who did not undergo resection (261 tumor slices) for survival group prediction. RESULTS: In both datasets, we show that habitat-based features achieved 87.50% and 86.36% accuracies for survival group prediction, respectively, using leave-one-out cross-validation. Experimental results revealed that spatially correlated features between signal-enhanced subregions were effective for predicting survival groups (P < 0.05 for all three machine-learning classifiers). CONCLUSION: The quantitative spatial-correlated features derived from MRI-defined tumor subregions in GBM could be effectively used to predict the survival time of patients. LEVEL OF EVIDENCE: 2 J. MAGN. RESON. IMAGING 2017;46:115-123.

Heterogeneity in intratumoral regions with rapid gadolinium washout correlates with estrogen receptor status and nodal metastasis.

PURPOSE: To evaluate heterogeneity within tumor subregions or "habitats" via textural kinetic analysis on breast dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for the classification of two clinical prognostic features; 1) estrogen receptor (ER)-positive from ER-negative tumors, and 2) tumors with four or more viable lymph node metastases after neoadjuvant chemotherapy from tumors without nodal metastases. MATERIALS AND METHODS: Two separate volumetric DCE-MRI datasets were obtained at 1.5T, comprised of bilateral axial dynamic 3D T1 -weighted fat suppressed gradient recalled echo-pulse sequences obtained before and after gadolinium-based contrast administration. Representative image slices of breast tumors from 38 and 34 patients were used for ER status and lymph node classification, respectively. Four tumor habitats were defined based on their kinetic contrast enhancement characteristics. The heterogeneity within each habitat was quantified using textural kinetic features, which were evaluated using two feature selectors and three classifiers. RESULTS: Textural kinetic features from the habitat with rapid delayed washout yielded classification accuracies of 84.44% (area under the curve [AUC] 0.83) for ER and 88.89% (AUC 0.88) for lymph node status. The texture feature, information measure of correlation, most often chosen in cross-validations, measures heterogeneity and provides accuracy approximately the same as with the best feature set. CONCLUSION: Heterogeneity within habitats with rapid washout is highly predictive of molecular tumor characteristics and clinical behavior.

Accelerating Fuzzy-C Means Using an Estimated Subsample Size.

Many algorithms designed to accelerate the Fuzzy c-Means (FCM) clustering algorithm randomly sample the data. Typically, no statistical method is used to estimate the subsample size, despite the impact subsample sizes have on speed and quality. This paper introduces two new accelerated algorithms, GOFCM and MSERFCM, that use a statistical method to estimate the subsample size. GOFCM, a variant of SPFCM, also leverages progressive sampling. MSERFCM, a variant of rseFCM, gains a speedup from improved initialization. A general, novel stopping criterion for accelerated clustering is introduced. The new algorithms are compared to FCM and four accelerated variants of FCM. GOFCM's speedup was 4-47 times that of FCM and faster than SPFCM on each of the six datasets used in experiments. For five of the datasets, partitions were within 1% of those of FCM. MSERFCM's speedup was 5-26 times that of FCM and produced partitions within 3% of those of FCM on all datasets. A unique dataset, consisting of plankton images, exposed the strengths and weaknesses of many of the algorithms tested. It is shown that the new stopping criterion is effective in speeding up algorithms such as SPFCM and the final partitions are very close to those of FCM.

Test-retest reproducibility analysis of lung CT image features.

Quantitative size, shape, and texture features derived from computed tomographic (CT) images may be useful as predictive, prognostic, or response biomarkers in non-small cell lung cancer (NSCLC). However, to be useful, such features must be reproducible, non-redundant, and have a large dynamic range. We developed a set of quantitative three-dimensional (3D) features to describe segmented tumors and evaluated their reproducibility to select features with high potential to have prognostic utility. Thirty-two patients with NSCLC were subjected to unenhanced thoracic CT scans acquired within 15 min of each other under an approved protocol. Primary lung cancer lesions were segmented using semi-automatic 3D region growing algorithms. Following segmentation, 219 quantitative 3D features were extracted from each lesion, corresponding to size, shape, and texture, including features in transformed spaces (laws, wavelets). The most informative features were selected using the concordance correlation coefficient across test-retest, the biological range and a feature independence measure. There were 66 (30.14%) features with concordance correlation coefficient ≥ 0.90 across test-retest and acceptable dynamic range. Of these, 42 features were non-redundant after grouping features with R (2) Bet ≥ 0.95. These reproducible features were found to be predictive of radiological prognosis. The area under the curve (AUC) was 91% for a size-based feature and 92% for the texture features (runlength, laws). We tested the ability of image features to predict a radiological prognostic score on an independent NSCLC (39 adenocarcinoma) samples, the AUC for texture features (runlength emphasis, energy) was 0.84 while the conventional size-based features (volume, longest diameter) was 0.80. Test-retest and correlation analyses have identified non-redundant CT image features with both high intra-patient reproducibility and inter-patient biological range. Thus making the case that quantitative image features are informative and prognostic biomarkers for NSCLC.

A novel deep learning-based method for automatic stereology of microglia cells from low magnification images.

Microglial cells mediate diverse homeostatic, inflammatory, and immune processes during normal development and in response to cytotoxic challenges. During these functional activities, microglial cells undergo distinct numerical and morphological changes in different tissue volumes in both rodent and human brains. However, it remains unclear how these cytostructural changes in microglia correlate with region-specific neurochemical functions. To better understand these relationships, neuroscientists need accurate, reproducible, and efficient methods for quantifying microglial cell number and morphologies in histological sections. To address this deficit, we developed a novel deep learning (DL)-based classification, stereology approach that links the appearance of Iba1 immunostained microglial cells at low magnification (20×) with the total number of cells in the same brain region based on unbiased stereology counts as ground truth. Once DL models are trained, total microglial cell numbers in specific regions of interest can be estimated and treatment groups predicted in a high-throughput manner (<1 min) using only low-power images from test cases, without the need for time and labor-intensive stereology counts or morphology ratings in test cases. Results for this DL-based automatic stereology approach on two datasets (total 39 mouse brains) showed >90% accuracy, 100% percent repeatability (Test-Retest) and 60× greater efficiency than manual stereology (<1 min vs. ∼ 60 min) using the same tissue sections. Ongoing and future work includes use of this DL-based approach to establish clear neurodegeneration profiles in age-related human neurological diseases and related animal models.

Automated Delineation of Lung Tumors from CT Images Using a Single Click Ensemble Segmentation Approach.

A single click ensemble segmentation (SCES) approach based on an existing "Click&Grow" algorithm is presented. The SCES approach requires only one operator selected seed point as compared with multiple operator inputs, which are typically needed. This facilitates processing large numbers of cases. Evaluation on a set of 129 CT lung tumor images using a similarity index (SI) was done. The average SI is above 93% using 20 different start seeds, showing stability. The average SI for 2 different readers was 79.53%. We then compared the SCES algorithm with the two readers, the level set algorithm and the skeleton graph cut algorithm obtaining an average SI of 78.29%, 77.72%, 63.77% and 63.76% respectively. We can conclude that the newly developed automatic lung lesion segmentation algorithm is stable, accurate and automated.

Experimental evaluation of baselines for forecasting social media timeseries.

Forecasting social media activity can be of practical use in many scenarios, from understanding trends, such as which topics are likely to engage more users in the coming week, to identifying unusual behavior, such as coordinated information operations or currency manipulation efforts. To evaluate a new approach to forecasting, it is important to have baselines against which to assess performance gains. We experimentally evaluate the performance of four baselines for forecasting activity in several social media datasets that record discussions related to three different geo-political contexts synchronously taking place on two different platforms, Twitter and YouTube. Experiments are done over hourly time periods. Our evaluation identifies the baselines which are most accurate for particular metrics and thus provides guidance for future work in social media modeling.

Modeling information diffusion in social media: data-driven observations.

Accurately modeling information diffusion within and across social media platforms has many practical applications, such as estimating the size of the audience exposed to a particular narrative or testing intervention techniques for addressing misinformation. However, it turns out that real data reveal phenomena that pose significant challenges to modeling: events in the physical world affect in varying ways conversations on different social media platforms; coordinated influence campaigns may swing discussions in unexpected directions; a platform's algorithms direct who sees which message, which affects in opaque ways how information spreads. This article describes our research efforts in the SocialSim program of the Defense Advanced Research Projects Agency. As formulated by DARPA, the intent of the SocialSim research program was "to develop innovative technologies for high-fidelity computational simulation of online social behavior ... [focused] specifically on information spread and evolution." In this article we document lessons we learned over the 4+ years of the recently concluded project. Our hope is that an accounting of our experience may prove useful to other researchers should they attempt a related project.

Ensembles of Convolutional Neural Networks for Survival Time Estimation of High-Grade Glioma Patients from Multimodal MRI.

Glioma is the most common type of primary malignant brain tumor. Accurate survival time prediction for glioma patients may positively impact treatment planning. In this paper, we develop an automatic survival time prediction tool for glioblastoma patients along with an effective solution to the limited availability of annotated medical imaging datasets. Ensembles of snapshots of three dimensional (3D) deep convolutional neural networks (CNN) are applied to Magnetic Resonance Image (MRI) data to predict survival time of high-grade glioma patients. Additionally, multi-sequence MRI images were used to enhance survival prediction performance. A novel way to leverage the potential of ensembles to overcome the limitation of labeled medical image availability is shown. This new classification method separates glioblastoma patients into long- and short-term survivors. The BraTS (Brain Tumor Image Segmentation) 2019 training dataset was used in this work. Each patient case consisted of three MRI sequences (T1CE, T2, and FLAIR). Our training set contained 163 cases while the test set included 46 cases. The best known prediction accuracy of 74% for this type of problem was achieved on the unseen test set.

A Review of Nuclei Detection and Segmentation on Microscopy Images Using Deep Learning With Applications to Unbiased Stereology Counting.

The detection and segmentation of stained cells and nuclei are essential prerequisites for subsequent quantitative research for many diseases. Recently, deep learning has shown strong performance in many computer vision problems, including solutions for medical image analysis. Furthermore, accurate stereological quantification of microscopic structures in stained tissue sections plays a critical role in understanding human diseases and developing safe and effective treatments. In this article, we review the most recent deep learning approaches for cell (nuclei) detection and segmentation in cancer and Alzheimer's disease with an emphasis on deep learning approaches combined with unbiased stereology. Major challenges include accurate and reproducible cell detection and segmentation of microscopic images from stained sections. Finally, we discuss potential improvements and future trends in deep learning applied to cell detection and segmentation.

A disector-based framework for the automatic optical fractionator.

Stereology-based methods provide the current state-of-the-art approaches for accurate quantification of numbers and other morphometric parameters of biological objects in stained tissue sections. The advent of artificial intelligence (AI)-based deep learning (DL) offers the possibility of improving throughput by automating the collection of stereology data. We have recently shown that DL can effectively achieve comparable accuracy to manual stereology but with higher repeatability, improved throughput, and less variation due to human factors by quantifying the total number of immunostained cells at their maximal profile of focus in extended depth of field (EDF) images. In the first of two novel contributions in this work, we propose a semi-automatic approach using a handcrafted Adaptive Segmentation Algorithm (ASA) to automatically generate ground truth on EDF images for training our deep learning (DL) models to automatically count cells using unbiased stereology methods. This update increases the amount of training data, thereby improving the accuracy and efficiency of automatic cell counting methods, without a requirement for extra expert time. The second contribution of this work is a Multi-channel Input and Multi-channel Output (MIMO) method using a U-Net deep learning architecture for automatic cell counting in a stack of z-axis images (also known as disector stacks). This DL-based digital automation of the ordinary optical fractionator ensures accurate counts through spatial separation of stained cells in the z-plane, thereby avoiding false negatives from overlapping cells in EDF images without the shortcomings of 3D and recurrent DL models. The contribution overcomes the issue of under-counting errors with EDF images due to overlapping cells in the z-plane (masking). We demonstrate the practical applications of these advances with automatic disector-based estimates of the total number of NeuN-immunostained neurons in a mouse neocortex. In summary, this work provides the first demonstration of automatic estimation of a total cell number in tissue sections using a combination of deep learning and the disector-based optical fractionator method.

Discovery of a Generalization Gap of Convolutional Neural Networks on COVID-19 X-Rays Classification.

A number of recent papers have shown experimental evidence that suggests it is possible to build highly accurate deep neural network models to detect COVID-19 from chest X-ray images. In this paper, we show that good generalization to unseen sources has not been achieved. Experiments with richer data sets than have previously been used show models have high accuracy on seen sources, but poor accuracy on unseen sources. The reason for the disparity is that the convolutional neural network model, which learns features, can focus on differences in X-ray machines or in positioning within the machines, for example. Any feature that a person would clearly rule out is called a confounding feature. Some of the models were trained on COVID-19 image data taken from publications, which may be different than raw images. Some data sets were of pediatric cases with pneumonia where COVID-19 chest X-rays are almost exclusively from adults, so lung size becomes a spurious feature that can be exploited. In this work, we have eliminated many confounding features by working with as close to raw data as possible. Still, deep learned models may leverage source specific confounders to differentiate COVID-19 from pneumonia preventing generalizing to new data sources (i.e. external sites). Our models have achieved an AUC of 1.00 on seen data sources but in the worst case only scored an AUC of 0.38 on unseen ones. This indicates that such models need further assessment/development before they can be broadly clinically deployed. An example of fine-tuning to improve performance at a new site is given.

An adaptive digital stain separation method for deep learning-based automatic cell profile counts.

BACKGROUND: Quantifying cells in a defined region of biological tissue is critical for many clinical and preclinical studies, especially in the fields of pathology, toxicology, cancer and behavior. As part of a program to develop accurate, precise and more efficient automatic approaches for quantifying morphometric changes in biological tissue, we have shown that both deep learning-based and hand-crafted algorithms can estimate the total number of histologically stained cells at their maximal profile of focus in Extended Depth of Field (EDF) images. Deep learning-based approaches show accuracy comparable to manual counts on EDF images but significant enhancement in reproducibility, throughput efficiency and reduced error from human factors. However, a majority of the automated counts are designed for single-immunostained tissue sections. NEW METHOD: To expand the automatic counting methods to more complex dual-staining protocols, we developed an adaptive method to separate stain color channels on images from tissue sections stained by a primary immunostain with secondary counterstain. COMPARISON WITH EXISTING METHODS: The proposed method overcomes the limitations of the state-of-the-art stain-separation methods, like the requirement of pure stain color basis as a prerequisite or stain color basis learning on each image. RESULTS: Experimental results are presented for automatic counts using deep learning-based and hand-crafted algorithms for sections immunostained for neurons (Neu-N) or microglial cells (Iba-1) with cresyl violet counterstain. CONCLUSION: Our findings show more accurate counts by deep learning methods compared to the handcrafted method. Thus, stain-separated images can function as input for automatic deep learning-based quantification methods designed for single-stained tissue sections.

Hybrid models for lung nodule malignancy prediction utilizing convolutional neural network ensembles and clinical data.

Purpose: Due to the high incidence and mortality rates of lung cancer worldwide, early detection of a precancerous lesion is essential. Low-dose computed tomography is a commonly used technique for screening, diagnosis, and prognosis of non-small-cell lung cancer. Recently, convolutional neural networks (CNN) had shown great potential in lung nodule classification. Clinical information (family history, gender, and smoking history) together with nodule size provide information about lung cancer risk. Large nodules have greater risk than small nodules. Approach: A subset of cases from the National Lung Screening Trial was chosen as a dataset in our study. We divided the nodules into large and small nodules based on different clinical guideline thresholds and then analyzed the groups individually. Similarly, we also analyzed clinical features by dividing them into groups. CNNs were designed and trained over each of these groups individually. To our knowledge, this is the first study to incorporate nodule size and clinical features for classification using CNN. We further made a hybrid model using an ensemble with the CNN models of clinical and size information to enhance malignancy prediction. Results: From our study, we obtained 0.9 AUC and 83.12% accuracy, which was a significant improvement over our previous best results. Conclusions: In conclusion, we found that dividing the nodules by size and clinical information for building predictive models resulted in improved malignancy predictions. Our analysis also showed that appropriately integrating clinical information and size groups could further improve risk prediction.

18F-FDG PET/CT Habitat Radiomics Predicts Outcome of Patients with Cervical Cancer Treated with Chemoradiotherapy.

PURPOSE: To determine if quantitative features extracted from pretherapy fluorine 18 fluorodeoxyglucose (18F-FDG) PET/CT estimate prognosis in patients with locally advanced cervical cancer treated with chemoradiotherapy. MATERIALS AND METHODS: In this retrospective study, PET/CT images and outcomes were curated from 154 patients with locally advanced cervical cancer, who underwent chemoradiotherapy from two institutions between March 2008 and June 2016, separated into independent training (n = 78; mean age, 51 years ± 13 [standard deviation]) and testing (n = 76; mean age, 50 years ± 10) cohorts. Radiomic features were extracted from PET, CT, and habitat (subregions with different metabolic characteristics) images that were derived by fusing PET and CT images. Parsimonious sets of these features were identified by the least absolute shrinkage and selection operator analysis and used to generate predictive radiomics signatures for progression-free survival (PFS) and overall survival (OS) estimation. Prognostic validation of the radiomic signatures as independent prognostic markers was performed using multivariable Cox regression, which was expressed as nomograms, together with other clinical risk factors. RESULTS: The radiomics nomograms constructed with T stage, lymph node status, and radiomics signatures resulted in significantly better performance for the estimation of PFS (Harrell concordance index [C-index], 0.85 for training and 0.82 for test) and OS (C-index, 0.86 for training and 0.82 for test) compared with International Federation of Gynecology and Obstetrics staging system (C-index for PFS, 0.70 for training [P = .001] and 0.70 for test [P = .002]; C-index for OS, 0.73 for training [P < .001] and 0.70 for test [P < .001]), respectively. CONCLUSION: Prognostic models were generated and validated from quantitative analysis of 18F-FDG PET/CT habitat images and clinical data, and may have the potential to identify the patients who need more aggressive treatment in clinical practice, pending further validation with larger prospective cohorts.Supplemental material is available for this article.© RSNA, 2020.

Lung Nodule Sizes Are Encoded When Scaling CT Image for CNN's.

Noninvasive diagnosis of lung cancer in early stages is one task where radiomics helps. Clinical practice shows that the size of a nodule has high predictive power for malignancy. In the literature, convolutional neural networks (CNNs) have become widely used in medical image analysis. We study the ability of a CNN to capture nodule size in computed tomography images after images are resized for CNN input. For our experiments, we used the National Lung Screening Trial data set. Nodules were labeled into 2 categories (small/large) based on the original size of a nodule. After all extracted patches were re-sampled into 100-by-100-pixel images, a CNN was able to successfully classify test nodules into small- and large-size groups with high accuracy. To show the generality of our discovery, we repeated size classification experiments using Common Objects in Context (COCO) data set. From the data set, we selected 3 categories of images, namely, bears, cats, and dogs. For all 3 categories a 5- × 2-fold cross-validation was performed to put them into small and large classes. The average area under receiver operating curve is 0.954, 0.952, and 0.979 for the bear, cat, and dog categories, respectively. Thus, camera image rescaling also enables a CNN to discover the size of an object. The source code for experiments with the COCO data set is publicly available in Github (https://github.com/VisionAI-USF/COCO_Size_Decoding/).

Deep Feature Stability Analysis Using CT Images of a Physical Phantom Across Scanner Manufacturers, Cartridges, Pixel Sizes, and Slice Thickness.

Image acquisition parameters for computed tomography scans such as slice thickness and field of view may vary depending on tumor size and site. Recent studies have shown that some radiomics features were dependent on voxel size (= pixel size × slice thickness), and with proper normalization, this voxel size dependency could be reduced. Deep features from a convolutional neural network (CNN) have shown great promise in characterizing cancers. However, how do these deep features vary with changes in imaging acquisition parameters? To analyze the variability of deep features, a physical radiomics phantom with 10 different material cartridges was scanned on 8 different scanners. We assessed scans from 3 different cartridges (rubber, dense cork, and normal cork). Deep features from the penultimate layer of the CNN before (pre-rectified linear unit) and after (post-rectified linear unit) applying the rectified linear unit activation function were extracted from a pre-trained CNN using transfer learning. We studied both the interscanner and intrascanner dependency of deep features and also the deep features' dependency over the 3 cartridges. We found some deep features were dependent on pixel size and that, with appropriate normalization, this dependency could be reduced. False discovery rate was applied for multiple comparisons, to mitigate potentially optimistic results. We also used stable deep features for prognostic analysis on 1 non-small cell lung cancer data set.

A Scalable Framework For Cluster Ensembles.

An ensemble of clustering solutions or partitions may be generated for a number of reasons. If the data set is very large, clustering may be done on tractable size disjoint subsets. The data may be distributed at different sites for which a distributed clustering solution with a final merging of partitions is a natural fit. In this paper, two new approaches to combining partitions, represented by sets of cluster centers, are introduced. The advantage of these approaches is that they provide a final partition of data that is comparable to the best existing approaches, yet scale to extremely large data sets. They can be 100,000 times faster while using much less memory. The new algorithms are compared against the best existing cluster ensemble merging approaches, clustering all the data at once and a clustering algorithm designed for very large data sets. The comparison is done for fuzzy and hard k-means based clustering algorithms. It is shown that the centroid-based ensemble merging algorithms presented here generate partitions of quality comparable to the best label vector approach or clustering all the data at once, while providing very large speedups.

Automatic ground truth for deep learning stereology of immunostained neurons and microglia in mouse neocortex.

Collection of unbiased stereology data currently relies on relatively simple, low throughput technology developed in the mid-1990s. In an effort to improve the accuracy and efficiency of these integrated hardware-software-digital microscopy systems, we have developed an automatic segmentation algorithm (ASA) for automatic stereology counts using the unbiased optical fractionator method. Here we report on a series of validation experiments in which immunostained neurons (NeuN) and microglia (Iba1) were automatically counted in tissue sections through a mouse neocortex. In the first step, a minimum of 100 systematic-random z-axis image stacks (disector stacks) containing NeuN- and Iba1-immunostained cells were automatically collected using a software-controlled 3 axes (XYZ) stage motor. In the second step, each disector stack was converted to an extended depth of field (EDF) image in which each cell is shown at its optimal plane of focus. Third, individual neurons and microglia were segmented and the regional minimas were extracted and used as seed regions for cells in a watershed segmentation algorithm. Finally, the unbiased disector frame and counting rules were used to make unbiased parameter estimates for neurons and microglia cells. The results for both NeuN neurons and Iba1 microglia were compared to manual counts made by a moderately experienced data collector from the same disector stacks. The final results show lower error rates for counts of Iba1-immunostained microglia cells than for quantifying NeuN-immunostained neurons, most likely due to less three-dimensional overlapping of Iba1 cells. We report that the throughput efficiency of using ASA to automatically annotate images of Iba1 microglia is more than five times greater than that of manual stereology counts of the same sections. Moreover, we show that ASA is significantly more accurate in counting microglia cells than a moderately experienced data collector (about 10% higher overall accuracy) when both were compared to counts by an expert neurohistologist. Thus, the ASA method applied to EDF images from disector stacks can be extremely useful to automate and increase the accuracy of cell counts, which could be especially helpful and cost-effective when expert help is not available. Another potential use of our ASA approach is to generate unsupervised ground truth as an efficient alternative to manual annotation for training deep learning models, as shown in our ongoing work.

Automatic stereology of mean nuclear size of neurons using an active contour framework.

The use of unbiased stereology to quantify structural parameters such as mean cell and nuclear size (area and volume) can be useful for a wide variety of biological studies. Here we propose a novel segmentation framework using an Active Contour Model to automate the collection of stereology from stained cells and other objects in tissue sections. This approach is demonstrated for stained brain sections from young adult Fischer 344 rats. Animals were perfused in-vivo with 4% paraformaldehyde and sectioned by frozen microtomy at an instrument setting of 40 µm. For each rat brain, a systematic-random set of sections through the entire substantia nigra pars compacta (SN) were immunostained to reveal tyrosine hydroxylase (TH)-immunopositive neurons. The novel framework applied an active contour (modified balloon snake) model with non-constant balloon force to automatically segment and quantify neuronal cell bodies by stereological point counting (SPC). Several contours were initialized in the image and based on the contour fit after 200 iterations classified as immunopositive (signal) or background contours in a sequential manner. Cell contours were determined in four steps based on several criteria, e.g., area of contour, dispersion measure, and degree of overlap. The image was automatically segmented according to the final contours. Using a point grid automatically generated at systematic-random orientations over the images, points hitting the segmented neural cell bodies were automatically counted. The final values from the automatic framework were compared with findings for ground truth (manual SPC). The results of this study show a strong agreement between data collected by the automatic framework and the ground truth (R2 ≥ 0.95) with a 5× gain in time efficiency for the automatic SPC. These findings give strong support for future applications of pattern recognition for assessing stereological parameters of biological objects identified by high signal:noise stains.