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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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.
The authors declare no competing financial interests.
Integrated supplementary information
The green beam path represents the excitation path and the red represents the emission path.
Numbers correspond to assembly flow as indicated in Steps 1 – 3.
Numbers correspond to assembly flow as indicated in Steps 4 – 7.
Example of using (a) bullseye spirit level [Step 8] and (b) tubular spirit level [Step 11] to align components.
Numbers correspond to assembly flow as indicated in Step 9A.
Supplementary Figure 6 Dichroic cube mount components and assembly process for epifluorescence imaging feature.
Numbers correspond to assembly flow as indicated in Step 9B.
Numbers correspond to assembly flow as indicated in Steps 10 – 16.
The base of the objective lens (above the treaded section) should be resting on the tube lens mount. Once the objective lens is properly secured to the stage mount, shift the lens mount up and down to ensure that the lens threading does not rub against the mount.
Numbers correspond to assembly flow as indicated in Steps 17 – 26.
(a) Two pairs of 1” x 3” glass slides are bonded to each other by applying superglue on the areas shown by blue arrows. Apply enough superglue so that the entire area of the base is covered. (b) Bond two additional glass slides on top and bottom of the two slide pairs and (c) wait until the glue has cured. (d) Mount the completed glass stage on the XY translation adapter, and tightly position it by sliding and locking the slider. The glass stage can then be secured by tightening four screws on the top of the XY translation adapter.
Numbers correspond to assembly flow as indicated in Step 27A.
Numbers correspond to assembly flow as indicated in Step 27B.
Supplementary Figure 13 Schematic diagram showing lens and diaphragm location for Köhler illumination.
Beam path showing the approximate location of lenses and iris diaphragms, where f is the focal length and fb is the back focal length of the lens. The actual positions may vary during the setup; hence calibration must be performed to achieve good Köhler illumination. For example, the first iris diaphragm (condenser diaphragm) must be located where the LED source is clearly visible after the condenser lens.
To achieve the desired magnifying power, (a) acquire an image of a calibration grid and measure the grid length (in pixels) with ImageJ. The effective magnification power is given by MLLM =detector pixel size [μm/px] /(grid size [μm] /measured number of pixels [px]). (b) If needed, adjust camera position. Repeat steps if necessary.
Supplementary Figure 15 Imaging area comparison with and without epifluorescence illumination add-on.
(a) Micrographs of 100 μm spaced target grid captured with and epi-fluorescence illumination module. Significant vignetting can be observed. (b) In comparison, the imaging area is larger if the epi-fluorescence module is omitted. Scale bar, 100 μm.
Analog scintillation images captured with (a) no binning, 1200× EM gain, and 1 min exposure time and (b) 4 binning, ~700× EM gain, and 2 min exposure time. (c) Digitial scintillation image captured with 4 binning, 1200× EM gain, 30 ms exposure time per frame, and 20,000 frames. Hot spots (red arrows) are caused by stray ionizing radiation directly hitting the EMCCD detector, frequently appears in analog images, whereas such signals are rejected in digitally reconstructed images. Scale bar, 200 μm.
Brightfield, fluorescence, bioluminescence, and overlay images from MDA-MB-231/Luc cells. Top row. Nikon 20x objective lens (effective magnification of 4x). Scale bar, 200 μm. Bottom row. Olympus 40x oil-immersion objective lens (effective magnification of ~11x). Scale bar, 100 μm.
Supplementary Figure 18 Example of bioluminescence imaging captured under short exposure times for real-time imaging.
(a) Brightfield image of MDA-MB-231/Luc cells and its corresponding bioluminescence images captured with different imaging parameters: (b) no binning, ~1060× EM gain, and 20 s exposure time; (c) no binning, 1200× EM gain, and 3 s exposure time; (d) 2 binning, 1200× EM gain, and 0.75 s exposure time. Scale bar, 200 μm.
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Kim, T., Türkcan, S. & Pratx, G. Modular low-light microscope for imaging cellular bioluminescence and radioluminescence. Nat Protoc 12, 1055–1076 (2017). https://doi.org/10.1038/nprot.2017.008
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