Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format
| Autor: | Benjamin T. Wilks, Jeffrey R. Morgan, Elizabeth Leary, Claire Rhee |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Microscope Materials science Microscopy Confocal Confocal Spheroid Article Computer Science Applications law.invention High-Throughput Screening Assays 03 medical and health sciences Medical Laboratory Technology 030104 developmental biology 0302 clinical medicine Organ Culture Techniques Confocal imaging law Cell Line Tumor Spheroids Cellular embryonic structures Humans Throughput (business) 030217 neurology & neurosurgery Biomedical engineering Fluorescent Dyes |
| Zdroj: | SLAS technology. 23(3) |
| ISSN: | 2472-6311 |
| Popis: | Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids. |
| Databáze: | OpenAIRE |
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