RESUMO
BACKGROUND AND OBJECTIVE: Cell segmentation in bright-field histological slides is a crucial topic in medical image analysis. Having access to accurate segmentation allows researchers to examine the relationship between cellular morphology and clinical observations. Unfortunately, most segmentation methods known today are limited to nuclei and cannot segment the cytoplasm. METHODS: We present a new network architecture Cyto R-CNN that is able to accurately segment whole cells (with both the nucleus and the cytoplasm) in bright-field images. We also present a new dataset CytoNuke, consisting of multiple thousand manual annotations of head and neck squamous cell carcinoma cells. Utilizing this dataset, we compared the performance of Cyto R-CNN to other popular cell segmentation algorithms, including QuPath's built-in algorithm, StarDist, Cellpose and a multi-scale Attention Deeplabv3+. To evaluate segmentation performance, we calculated AP50, AP75 and measured 17 morphological and staining-related features for all detected cells. We compared these measurements to the gold standard of manual segmentation using the Kolmogorov-Smirnov test. RESULTS: Cyto R-CNN achieved an AP50 of 58.65% and an AP75 of 11.56% in whole-cell segmentation, outperforming all other methods (QuPath 19.46/0.91%; StarDist 45.33/2.32%; Cellpose 31.85/5.61%, Deeplabv3+ 3.97/1.01%). Cell features derived from Cyto R-CNN showed the best agreement to the gold standard (D¯=0.15) outperforming QuPath (D¯=0.22), StarDist (D¯=0.25), Cellpose (D¯=0.23) and Deeplabv3+ (D¯=0.33). CONCLUSION: Our newly proposed Cyto R-CNN architecture outperforms current algorithms in whole-cell segmentation while providing more reliable cell measurements than any other model. This could improve digital pathology workflows, potentially leading to improved diagnosis. Moreover, our published dataset can be used to develop further models in the future.
Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador , Redes Neurais de Computação , Humanos , Processamento de Imagem Assistida por Computador/métodos , Núcleo Celular , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Citoplasma , Reprodutibilidade dos Testes , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologiaRESUMO
Penile, vulvar and anal neoplasms show an incidence lower than 0.5% of the population per year and therefore can be considered as rare cancers but with a dramatic impact on quality of life and survival. This work describes the experience of a Chilean cancer center using multiplexed immunofluorescence to study a case series of four penile cancers, two anal cancers and one vulvar cancer and simultaneous detection of CD8, CD68, PD-L1, Cytokeratin and Ki-67 in FFPE samples. Fluorescent image analyses were performed using open sources for automated tissue segmentation and cell phenotyping. Our results showed an objective and reliable counting of objects with a single or combined labeling or within a specific tissue compartment. The variability was below 10%, and the correlation between analytical events was 0.92-0.97. Critical cell phenotypes, such as TILs, PD-L1+ or proliferative tumor cells were detected in a supervised and unsupervised manner with a limit of detection of less than 1% of relative abundance. Finally, the observed diversity and abundance of the different cell phenotypes within the tumor microenvironment for the three studied tumor types confirmed that our methodology is useful and robust to be applicable for many other solid tumors.
RESUMO
The development of advanced techniques in medical imaging has allowed scanning of the human body to microscopic levels, making research on cell behavior more complex and more in-depth. Recent studies have focused on cellular heterogeneity since cell-to-cell differences are always present in the cell population and this variability contains valuable information. However, identifying each cell is not an easy task because, in the images acquired from the microscope, there are clusters of cells that are touching one another. Therefore, the segmentation stage is a problem of considerable difficulty in cell image processing. Although several methods for cell segmentation are described in the literature, they have drawbacks in terms of over-segmentation, under-segmentation or misidentification. Consequently, our main motivation in studying cell segmentation was to develop a new method to achieve a good tradeoff between accurately identifying all relevant elements and not inserting segmentation artifacts. This article presents a new method for cell segmentation in fluorescence microscopy images. The proposed approach combines the well-known Marker-Controlled Watershed algorithm (MC-Watershed) with a new, two-step method based on Watershed, Split and Merge Watershed (SM-Watershed): in the first step, or split phase, the algorithm identifies the clusters using inherent characteristics of the cell, such as size and convexity, and separates them using watershed. In the second step, or the merge stage, it identifies the over-segmented regions using proper features of the cells and eliminates the divisions. Before applying our two-step method, the input image is first preprocessed, and the MC-Watershed algorithm is used to generate an initial segmented image. However, this initial result may not be suitable for subsequent tasks, such as cell count or feature extraction, because not all cells are separated, and some cells may be mistakenly confused with the background. Thus, our proposal corrects this issue with its two-step process, reaching a high performance, a suitable tradeoff between over-segmentation and under-segmentation and preserving the shape of the cell, without the need of any labeled data or relying on machine learning processes. The latter is advantageous over state-of-the-art techniques that in order to achieve similar results require labeled data, which may not be available for all of the domains. Two cell datasets were used to validate this approach, and the results were compared with other methods in the literature, using traditional metrics and quality visual assessment. We obtained 90% of average visual accuracy and an F-index higher than 80%. This proposal outperforms other techniques for cell separation, achieving an acceptable balance between over-segmentation and under-segmentation, which makes it suitable for several applications in cell identification, such as virus infection analysis, high-content cell screening, drug discovery, and morphometry.
RESUMO
Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.