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Imaging liver biology in vivo using conventional confocal microscopy

Nature Protocols volume 10, pages 258–268 (2015)Cite this article

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Abstract

Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.

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Figure 1: Conventional microscope setup for confocal intravital microscopy.
Figure 2: Detailed surgical procedure for liver imaging using confocal intravital microscopy.
Figure 3: Multichannel in vivo imaging.
Figure 4: Investigating cell death and release of intracellular contents in vivo.
Figure 5: Microbead arrest by Kupffer cells visualized by confocal intravital microscopy.
Figure 6: Assessment of in vivo neutrophil migration to necrotic sites using liver intravital microscopy.
Figure 7: 4D rendering of liver intravital microscopy: Snapshot showing in vivo neutrophil migration to necrotic sites.

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Acknowledgements

We thank L. Chang and E.M. Nishitani for their help in figure preparation, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais/Programa de Apoio aos Núcleos de Excelência (FAPEMIG/PRONEX) for their financial support.

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Author notes

  1. Pedro E Marques and Maísa M Antunes: These authors contributed equally to this work.

Authors and Affiliations

  1. Departamento de Morfologia, Laboratório de Imunobiofotônica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.,

    Pedro E Marques, Maísa M Antunes, Bruna A David, Rafaela V Pereira & Gustavo B Menezes

  2. Departamento de Bioquímica e Imunologia, Imunofarmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.,

    Mauro M Teixeira

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  1. Pedro E Marques
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Contributions

P.E.M., M.M.A., B.A.D., R.V.P. and G.B.M. designed the method and performed experiments to generate data presented as anticipated results; and P.E.M., M.M.A., M.M.T. and G.B.M. wrote and revised the manuscript.

Corresponding author

Correspondence to Gustavo B Menezes.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Control experiments using FITC and PE-conjugated isotype controls.

In these experiments, mice received acetaminophen (APAP) via gavage (600 mg/kg; 24 h before imaging) to induce acute liver injury. Using the same mouse, both isotype controls were intravenously injected in a mixture. Note that no significant stain is observed. Sequentially, anti-F4/80 (in red) and anti-Ly-6G were administrated in the same route. Note that now, Kupffer cells and neutrophils were visualized in the liver microvasculature of APAP-treated mouse, confirming antibody specificity. Microscope acquisition settings: 488 nm - laser power: 10.2%; PMT HV: 82; 543 nm - laser power: 41,3%; PMT HV: 103. Pinhole size: 40 μm. 10x objective. Bar = 100 μm. Appropriate Institutional Regulatory Board permission for animal use was obtained.

Supplementary Figure 2 Custom-made Plexiglas stage to image living small rodents (mice and young rats) under a confocal microscope.

This stage is made in the provided sizes in order to adapt in the most slide holders commercially available. Size adjustments might be necessary. Note that the complete sealing of the borders will avoid superfusion buffer leakage to the microscope and unwanted damages.

Supplementary Figure 3 Snapshot of the acquisition screen from NIS Elements version 4.20.

(a) Note that separated screens may provide better visualization during acquisition, and (b) improving the correct control for acquisition functions.

Supplementary Figure 4 Additional procedures to generate liver focal thermal injury

In a, note that a 30G or sewing needle was adapted to the cauterizer tip to act as a thermal probe to create a small, localized necrotic area in the liver. (b) This was made by gently touch the liver surface with a previously heated needle. Appropriate Institutional Regulatory Board permission for animal use was obtained.

Supplementary Figure 5 Continuous imaging procedure for photobleaching investigation.

Mice received acetaminophen (APAP) via gavage (600 mg/kg; 12 h before imaging) to induce acute liver injury. Note a widespread DNA accumulation stained by Sytox green within the liver. No significant photobleaching is observed during laser exposure for 20 minutes. However, DNA derived fluorescence is faded immediately after intravenous DNASE1 injection (1000 U/mouse). Fluorescence mean (in arbitrary units, AU) was estimated using Volocity software). See also Supplementary Video 6. Microscope acquisition settings: 488 nm - laser power: 5.8%; PMT HV: 87; Pinhole size: 40 μm. 10x objective. Bar = 100 μm. Appropriate Institutional Regulatory Board permission for animal use was obtained.

Supplementary Figure 6 Snapshot of a regular tracking protocol using Volocity.

A sequential inclusion/exclusion protocol will initially define which objects to be tracked. This step is crucial to avoid missing or unwanted tracks. Objects may be defined by their size, brightness, roundness or volume depending on the experimental protocol. Detailed tracking tutorials are provided in Volocity website (see Anticipated Results).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 2337 kb)

Detailed surgical procedure to perform liver intravital microscopy.

Description of surgical technique to expose the liver to be imaged under the confocal microscope. (See also Fig. 2). Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 25222 kb)

Time-lapse 3D rendering of neutrophils crawling to necrotic spots.

A punctual necrotic spot was generated using the tip of a hot needle adapted to a cauterizer pen. Dead cells were stained using propidium iodide (in red). Z-stacks were acquired for 4 hours. Video was accelerated to better visualization of neutrophils crawling. Note that the preparation is steady throughout all image procedure. Microscope acquisition settings: 488 nm - laser power: 5.8%; PMT HV: 75; 543 nm - laser power: 1.3%; PMT HV: 103. Pinhole size: 40 μm. 10× objective. Movie was acquired using 3 minutes interval between frames, and 66 loops were done. Z-steps were 2 μm thick and the total depth was 48 μm. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 1770 kb)

Three-channel liver intravital microscopy using Lysm-eGFP mouse (neutrophils in green).

Spontaneous intravascular neutrophil crawling was imaged for 10 minutes. Liver sinusoidal cells were evidenced by BV421-conjugated anti-CD31 (PECAM-1; in blue) and Kupffer cells were stained using PE-conjugated anti-F4/80. Video was accelerated to better visualization of neutrophil crawling. Microscope acquisition settings: 405 nm - laser power: 47.9%; PMT HV: 98; 488 nm - laser power: 17.6%; PMT HV: 80; 543 nm - laser power: 44.6%; PMT HV: 100. Pinhole size: 40 μm. 40× objective. Movie was acquired using no interval between frames, and 31 loops were done. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 3240 kb)

Acetaminophen-mediated injury caused DNA deposition in the liver: real-time DNA metabolism by intravenous DNASE1 injection.

Mice received acetaminophen (600 mg/kg) 12 h before imaging. Note that a spread hepatic DNA deposition (in green, Sytox green staining) is observed. Following 5 minutes of intravenous DNASE1 injection, DNA is rapidly removed from the circulation. Hepatocyte morphology was evidenced by rhodamine 6G injection (in red). Video was acquired for 10 minutes, with no delay between frames. A total of 70 loops were done. Video was accelerated to better visualization of DNA metabolism. Microscope acquisition settings: 488 nm - laser power: 17.7%; PMT HV: 90; 543 nm - laser power: 14.7%; PMT HV: 81. Pinhole size: 40 μm. 10× objective and 2.0× digital zoom. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 3242 kb)

Kupffer Imaging cells and their high phagocytic activity in vivo.

Kupffer cells were stained by PE-conjugated anti- F4/80 (in red). While recording, fluorescent microspheres (in green) were injected intravenously. Note that Kupffer cells avidly arrested several beads and no more free circulating microspheres were seen after 5-10 minutes. Video was acquired using an interval of 3 seconds between each frame. Video was accelerated to better visualization of bead arrest. Microscope acquisition settings: 488 nm - laser power: 4.2%; PMT HV: 52; 543 nm - laser power: 34.6%; PMT HV: 117. Pinhole size: 40 μm. 10× objective. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 5371 kb)

Continuous imaging procedure for photobleaching investigation.

Mice received acetaminophen (APAP) via gavage (600 mg/kg; 12 h before imaging) to induce acute liver injury. Note a widespread DNA accumulation stained by Sytox green within the liver. No significant photobleaching is observed during laser exposure for 20 minutes. However, DNA derived fluorescence is faded immediately after intravenous DNASE1 injection (1000 U/mouse). Microscope acquisition settings: 488 nm - laser power: 5.8%; PMT HV: 87; Pinhole size: 40 μm. 10× objective.61 loops were acquired. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 3823 kb)

Neutrophils precisely crawl to necrotic spots.

Liver intravital microscopy using Lysm-eGFP mouse (neutrophils in green) showing 4 hours of acquisition of the same region. A punctual necrotic spot was generated using the tip of a hot needle adapted to a cauterizer pen. Dead cells were stained using propidium iodide (in red). Video was acquired using an interval of 1 minute between each frame. Video was accelerated to better visualization of neutrophils crawling. Note that the preparation is steady throughout all image procedure. Microscope acquisition settings: 488 nm - laser power: 17.8%; PMT HV: 100; 543 nm - laser power: 5.0%; PMT HV: 93. Pinhole size: 40 μm. 10× objective. Movie was acquired using 1 minute interval between frames, and 241 loops were done. Appropriate Institutional Regulatory Board permission for animal use was obtained. (MOV 2792 kb)

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Marques, P., Antunes, M., David, B. et al. Imaging liver biology in vivo using conventional confocal microscopy. Nat Protoc 10, 258–268 (2015). https://doi.org/10.1038/nprot.2015.006

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