Optimizing the Cell Painting assay for image-based profilingCimini, Beth A.; Chandrasekaran, Srinivas Niranj; Kost-Alimova, Maria; Miller, Lisa; Goodale, Amy; Fritchman, Briana; Byrne, Patrick; Garg, Sakshi; Jamali, Nasim; Logan, David J.; Concannon, John B.; Lardeau, Charles-Hugues; Mouchet, Elizabeth; Singh, Shantanu; Shafqat Abbasi, Hamdah; Aspesi, Peter; Boyd, Justin D.; Gilbert, Tamara; Gnutt, David; Hariharan, Santosh; Hernandez, Desiree; Hormel, Gisela; Juhani, Karolina; Melanson, Michelle; Mervin, Lewis H.; Monteverde, Tiziana; Pilling, James E.; Skepner, Adam; Swalley, Susanne E.; Vrcic, Anita; Weisbart, Erin; Williams, Guy; Yu, Shan; Zapiec, Bolek; Carpenter, Anne E.
doi: 10.1038/s41596-023-00840-9pmid: 37344608
In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years’ experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay’s ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1–2 weeks.This protocol is an update to Nat. Protoc. 11, 1757–1774 (2016): https://doi.org/10.1038/nprot.2016.105
Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworksChen, Guosheng; Huang, Siming; Ma, Xiaomin; He, Rongwei; Ouyang, Gangfeng
doi: 10.1038/s41596-023-00828-5pmid: 37198321
Enzymes are outstanding natural catalysts with exquisite 3D structures, initiating countless life-sustaining biotransformations in living systems. The flexible structure of an enzyme, however, is highly susceptible to non-physiological environments, which greatly limits its large-scale industrial applications. Seeking suitable supports to immobilize fragile enzymes is one of the most efficient routes to ameliorate the stability problem. This protocol imparts a new bottom-up strategy for enzyme encapsulation using a hydrogen-bonded organic framework (HOF-101). In short, the surface residues of the enzyme can trigger the nucleation of HOF-101 around its surface through the hydrogen-bonded biointerface. As a result, a series of enzymes with different surface chemistries are able to be encapsulated within a highly crystalline HOF-101 scaffold, which has long-range ordered mesochannels. The details of experimental procedures are described in this protocol, which involve the encapsulating method, characterizations of materials and biocatalytic performance tests. Compared with other immobilization methods, this enzyme-triggering HOF-101 encapsulation is easy to operate and affords higher loading efficiency. The formed HOF-101 scaffold has an unambiguous structure and well-arranged mesochannels, favoring mass transfer and understanding of the biocatalytic process. It takes ~13.5 h for successful synthesis of enzyme-encapsulated HOF-101, 3–4 d for characterizations of materials and ~4 h for the biocatalytic performance tests. In addition, no specific expertise is necessary for the preparation of this biocomposite, although the high-resolution imaging requires a low-electron-dose microscope technology. This protocol can provide a useful methodology to efficiently encapsulate enzymes and design biocatalytic HOF materials.
Spatial- and Fourier-domain ptychography for high-throughput bio-imagingJiang, Shaowei; Song, Pengming; Wang, Tianbo; Yang, Liming; Wang, Ruihai; Guo, Chengfei; Feng, Bin; Maiden, Andrew; Zheng, Guoan
doi: 10.1038/s41596-023-00829-4pmid: 37248392
First envisioned for determining crystalline structures, ptychography has become a useful imaging tool for microscopists. However, ptychography remains underused by biomedical researchers due to its limited resolution and throughput in the visible light regime. Recent developments of spatial- and Fourier-domain ptychography have successfully addressed these issues and now offer the potential for high-resolution, high-throughput optical imaging with minimal hardware modifications to existing microscopy setups, often providing an excellent trade-off between resolution and field of view inherent to conventional imaging systems, giving biomedical researchers the best of both worlds. Here, we provide extensive information to enable the implementation of ptychography by biomedical researchers in the visible light regime. We first discuss the intrinsic connections between spatial-domain coded ptychography and Fourier ptychography. A step-by-step guide then provides the user instructions for developing both systems with practical examples. In the spatial-domain implementation, we explain how a large-scale, high-performance blood-cell lens can be made at negligible expense. In the Fourier-domain implementation, we explain how adding a low-cost light source to a regular microscope can improve the resolution beyond the limit of the objective lens. The turnkey operation of these setups is suitable for use by professional research laboratories, as well as citizen scientists. Users with basic experience in optics and programming can build the setups within a week. The do-it-yourself nature of the setups also allows these procedures to be implemented in laboratory courses related to Fourier optics, biomedical instrumentation, digital image processing, robotics and capstone projects.
Spiral volumetric optoacoustic tomography for imaging whole-body biodynamics in small animalsKalva, Sandeep Kumar; Deán-Ben, Xosé Luís; Reiss, Michael; Razansky, Daniel
doi: 10.1038/s41596-023-00834-7pmid: 37208409
Fast tracking of biological dynamics across multiple murine organs using the currently commercially available whole-body preclinical imaging systems is hindered by their limited contrast, sensitivity and spatial or temporal resolution. Spiral volumetric optoacoustic tomography (SVOT) provides optical contrast, with an unprecedented level of spatial and temporal resolution, by rapidly scanning a mouse using spherical arrays, thus overcoming the current limitations in whole-body imaging. The method enables the visualization of deep-seated structures in living mammalian tissues in the near-infrared spectral window, while further providing unrivalled image quality and rich spectroscopic optical contrast. Here, we describe the detailed procedures for SVOT imaging of mice and provide specific details on how to implement a SVOT system, including component selection, system arrangement and alignment, as well as the image processing methods. The step-by-step guide for the rapid panoramic (360°) head-to-tail whole-body imaging of a mouse includes the rapid visualization of contrast agent perfusion and biodistribution. The isotropic spatial resolution possible with SVOT can reach 90 µm in 3D, while alternative steps enable whole-body scans in less than 2 s, unattainable with other preclinical imaging modalities. The method further allows the real-time (100 frames per second) imaging of biodynamics at the whole-organ level. The multiscale imaging capacity provided by SVOT can be used for visualizing rapid biodynamics, monitoring responses to treatments and stimuli, tracking perfusion, and quantifying total body accumulation and clearance dynamics of molecular agents and drugs. Depending on the imaging procedure, the protocol requires 1–2 h to complete by users trained in animal handling and biomedical imaging.
Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assayLago, Chiara; Gianesello, Matteo; Santomaso, Lucia; Leva, Gloria; Ballabio, Claudio; Anderle, Marica; Antonica, Francesco; Tiberi, Luca
doi: 10.1038/s41596-023-00839-2pmid: 37248391
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60–65 d for cancer organoids generation and from 1–4 weeks for downstream applications. The protocol requires at least 3–6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag micede Mello, Natalia Prudente; Fecher, Caroline; Pastor, Adrian Marti; Perocchi, Fabiana; Misgeld, Thomas
doi: 10.1038/s41596-023-00831-wpmid: 37328604
Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse. While other available methods were developed for bulk mitochondria isolation or more abundant cell-type-specific mitochondria, this protocol was optimized for the selective isolation of functional mitochondria from medium-to-low-abundant cell types in a heterogeneous tissue, such as the central nervous system. The protocol has three major parts: First, mitochondria of a cell type of interest are tagged via an outer mitochondrial membrane eGFP by crossing MitoTag mice to a cell-type-specific Cre-driver line or by delivery of viral vectors for Cre expression. Second, homogenates are prepared from relevant tissues by nitrogen cavitation, from which tagged organelles are immunocaptured using magnetic microbeads. Third, immunocaptured mitochondria are used for downstream assays, e.g., to probe respiratory capacity or calcium handling, revealing cell-type-specific mitochondrial diversity in molecular composition and function. The MitoTag approach enables the identification of marker proteins to label cell-type-specific organelle populations in situ, elucidates cell-type-enriched mitochondrial metabolic and signaling pathways, and reveals functional mitochondrial diversity between adjacent cell types in complex tissues, such as the brain. Apart from establishing the mouse colony (6–8 weeks without import), the immunocapture protocol takes 2 h and functional assays require 1–2 h.
Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editorsLei, Zhixin; Meng, Haowei; Rao, Xichen; Zhao, Huanan; Yi, Chengqi
doi: 10.1038/s41596-023-00837-4pmid: 37277562
Programmable cytosine base editors show promising approaches for correcting pathogenic mutations; yet, their off-target effects have been of great concern. Detect-seq (dU-detection enabled by C-to-T transition during sequencing) is an unbiased, sensitive method for the off-target evaluation of programmable cytosine base editors. It profiles the editome by tracing the editing intermediate dU, which is introduced inside living cells and edited by programmable cytosine base editors. The genomic DNA is extracted, preprocessed and labeled by successive chemical and enzymatic reactions, followed by biotin pull-down to enrich the dU-containing loci for sequencing. Here, we describe a detailed protocol for performing the Detect-seq experiment, and a customized, open-source, bioinformatic pipeline for analyzing the characteristic Detect-seq data is also provided. Unlike those previous whole-genome sequencing-based methods, Detect-seq uses an enrichment strategy and hence is endowed with great sensitivity, a higher signal-to-noise ratio and no requirement for high sequencing depth. Furthermore, Detect-seq is widely applicable for both mitotic and postmitotic biological systems. The entire protocol typically takes 5 d from the genomic DNA extraction to sequencing and ~1 week for data analysis.