Protocol | Published:

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

Nature Protocols volume 12, pages 10551076 (2017) | Download Citation

Abstract

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1–4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as those of radioluminescence (1–1.5 h), bioluminescence (30 min) and low-excitation fluorescence (15 min). In particular, radioluminescence microscopy is explained in detail, as it is a newly developed technique that enables the study of small-molecule transport (e.g., radiolabeled drugs, metabolic precursors and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (e.g., CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution images of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed together on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level.

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Acknowledgements

This work was supported by funding from the National Institutes of Health (NIH) under grants 5R01CA186275 and 1R21CA193001. T.J.K. was supported in part by NCI training grant T32 CA118681. The authors gratefully acknowledge the support of Olympus for its loan of the LV200 microscope.

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Affiliations

  1. Department of Radiation Oncology, Division of Medical Physics, Stanford University School of Medicine, Palo Alto, California, USA.

    • Tae Jin Kim
    • , Silvan Türkcan
    •  & Guillem Pratx

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Contributions

T.J.K. designed and built the low-light microscope system; T.J.K. and G.P. developed the graphical user interface version of ORBIT; T.J.K. and S.T. prepared radiolabeled samples; T.J.K. performed experiments and analyzed data; T.J.K. and G.P. prepared the manuscript; S.T. provided manuscript feedback; and G.P. provided guidance for the entire project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Guillem Pratx.

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https://doi.org/10.1038/nprot.2017.008

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