Correlative Fluorescence and Raman Microscopy to Define Mitotic Stages at the Single-Cell Level: Opportunities and Limitations in the AI Era.

Nowadays, morphology and molecular analyses at the single-cell level have a fundamental role in understanding biology better. These methods are utilized for cell phenotyping and in-depth studies of cellular processes, such as mitosis. Fluorescence microscopy and optical spectroscopy techniques, including Raman micro-spectroscopy, allow researchers to examine biological samples at the single-cell level in a non-destructive manner. Fluorescence microscopy can give detailed morphological information about the localization of stained molecules, while Raman microscopy can produce label-free images at the subcellular level; thus, it can reveal the spatial distribution of molecular fingerprints, even in live samples. Accordingly, the combination of correlative fluorescence and Raman microscopy (CFRM) offers a unique approach for studying cellular stages at the single-cell level. However, subcellular spectral maps are complex and challenging to interpret. Artificial intelligence (AI) may serve as a valuable solution to characterize the molecular backgrounds of phenotypes and biological processes by finding the characteristic patterns in spectral maps. The major contributions of the manuscript are: (I) it gives a comprehensive review of the literature focusing on AI techniques in Raman-based cellular phenotyping; (II) via the presentation of a case study, a new neural network-based approach is described, and the opportunities and limitations of AI, specifically deep learning, are discussed regarding the analysis of Raman spectroscopy data to classify mitotic cellular stages based on their spectral maps.

nucleAIzer: A Parameter-free Deep Learning Framework for Nucleus Segmentation Using Image Style Transfer.

Single-cell segmentation is typically a crucial task of image-based cellular analysis. We present nucleAIzer, a deep-learning approach aiming toward a truly general method for localizing 2D cell nuclei across a diverse range of assays and light microscopy modalities. We outperform the 739 methods submitted to the 2018 Data Science Bowl on images representing a variety of realistic conditions, some of which were not represented in the training data. The key to our approach is that during training nucleAIzer automatically adapts its nucleus-style model to unseen and unlabeled data using image style transfer to automatically generate augmented training samples. This allows the model to recognize nuclei in new and different experiments efficiently without requiring expert annotations, making deep learning for nucleus segmentation fairly simple and labor free for most biological light microscopy experiments. It can also be used online, integrated into CellProfiler and freely downloaded at www.nucleaizer.org. A record of this paper's transparent peer review process is included in the Supplemental Information.

A Deep Learning-Based Approach for High-Throughput Hypocotyl Phenotyping.

Hypocotyl length determination is a widely used method to phenotype young seedlings. The measurement itself has advanced from using rulers and millimeter papers to assessing digitized images but remains a labor-intensive, monotonous, and time-consuming procedure. To make high-throughput plant phenotyping possible, we developed a deep-learning-based approach to simplify and accelerate this method. Our pipeline does not require a specialized imaging system but works well with low-quality images produced with a simple flatbed scanner or a smartphone camera. Moreover, it is easily adaptable for a diverse range of datasets not restricted to Arabidopsis (Arabidopsis thaliana). Furthermore, we show that the accuracy of the method reaches human performance. We not only provide the full code at https://github.com/biomag-lab/hypocotyl-UNet, but also give detailed instructions on how the algorithm can be trained with custom data, tailoring it for the requirements and imaging setup of the user.

Phenotypic Image Analysis Software Tools for Exploring and Understanding Big Image Data from Cell-Based Assays.

Phenotypic image analysis is the task of recognizing variations in cell properties using microscopic image data. These variations, produced through a complex web of interactions between genes and the environment, may hold the key to uncover important biological phenomena or to understand the response to a drug candidate. Today, phenotypic analysis is rarely performed completely by hand. The abundance of high-dimensional image data produced by modern high-throughput microscopes necessitates computational solutions. Over the past decade, a number of software tools have been developed to address this need. They use statistical learning methods to infer relationships between a cell's phenotype and data from the image. In this review, we examine the strengths and weaknesses of non-commercial phenotypic image analysis software, cover recent developments in the field, identify challenges, and give a perspective on future possibilities.