Single-Cell Live Imaging
Recent fluorescence microscopy allows for high-throughput acquisition of 5D (X, Y, Z, T, and Color) images in various targets such as cultured cells, 3D spheroid/organoid, and even living tissue with single-cell resolution. The technology is considered pr
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Introduction Since the development of fluorescence microscopy, the technology has continuously improved its fundamental features, such as resolution, speed, and time-lapse image acquisition. Simultaneously, development of various fluorescent protein mutants and probes significantly accelerated its applications. Fluorescence microscopy has now become an essential tool for a wide range of biological and medical studies. Single cell live imaging allows for time-series understanding of individual cells while maintaining their culture or living conditions. This represents significant advantage over other population-based methods such as Western blotting and immunohistochemistry of fixed samples. Live imaging of cultured cells with epifluorescence microscopy now allows for automated observation of multiple wells such as 96 wells, which especially benefits drug screening and multitarget siRNA screening studies. On the other hand, the application of new microscopy such as multiphoton excitation microscopy has been utilized to capture in vivo dynamics of cells in living organisms from Drosophila
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_24, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Toru Hiratsuka and Naoki Komatsu
melanogaster, Caenorhabditis elegans, zebrafish, mouse, and even living human skin [1, 2]. Here we describe general protocols for single cell imaging of 2D-cultured cells with epifluorescence microscopy, 3D-cultured spheroid/organoid with confocal microscopy, and living mouse tissue with multiphoton microscopy. For more details, we exemplify the technology with single cell timelapse FRET imaging of cultured cells (2D) and single cell mouse skin tissue imaging (3D).
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Materials
2.1 General Equipment
1. Microscope systems (including objective lens, detectors, and PC installed with microscope control software) (see Note 1). 2. CO2 stage incubator. 3. Heating chamber. 4. Sample dish/plate holder for microscope stage. 5. Phenol-red free imaging media. 6. (If nuclear labeling is needed) Hoechst 33342. 7. Image analysis software (see Note 2). 8. PC for image analysis.
2.2 Cell Culture Reagents
1. Collagen type I-C solution. 2. Expression plasmid for fluorescent probe. 3. Transfection reagents (e.g., Lipofection reagents).
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Mouse Imaging
1. Depilation cream. 2. Pulse oximeter. 3. Animal temperature controller. 4. Anesthesia machine. 5. Heat pad for mouse.
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Methods
3.1 Sample Preparation 3.1.1 2D-Cultured Cells
Plate cells onto a glass-based dish/well (0.15–0.18 μm of thickness). Coating the glass with extracellular matrices such as collagen and poly-L-lysine promotes cell attachment (see Note 3). After complete attachment (1 day), transfect the cells with fluorescence expression plasmids by lipofection reagents (see Note 4). Incubate cells under 37 C humidified CO2 incubator for 1–2 days (see Note 5) to allow cells to express sufficient amount of
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