Simplifying Experiments Involving CLEM with a Fluorescence Microscope

Correlative light and electron microscopy (CLEM) is a robust method for examining cellular processes on an ultrastructural level. The SECOM platform, a fluorescence microscope developed to be combined with a scanning electron microscope, is capable of streamlining experiments involving CLEM by providing an entirely automated overlay procedure and smooth switching between fluorescence and electron microscopy.

Challenge

Sample preparation, however, is still the core of any experiment. To attain the ideal overlay accuracy, one preferably obtains the fluorescence as well as the electron images on the same resin-embedded section [1, 2]. This gives rise to a difficulty in relation to sample preparation. Luckily, latest advances in sample preparation have resulted in a range of protocols that show in-resin fluorescence of fluorescent proteins [1-5].

Two studies reveal that imaging GFP and YFP in vacuum has a large impact on the fluorescent intensity. From [3]: “First, we observed a drop in fluorescence intensity as the vacuum pressure decreases due to extraction of water from the sample, and which can be reversed by re-introducing water into the system at partial vacuum pressure or atmospheric pressure. Second, we show that although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable and resistant to photobleaching during imaging. Finally, we show that holding IRF sections in vacuum leads to very minor losses in fluorescence over time.”

Experiment

In this case, Hela Kyoto cells were used, which stably express GalNAC-T2-GFP and Histone 2B-mcherry. The cells were grown on carbon-coated sapphire disks and frozen at high pressure. Then, the cells were freeze replaced with 0.1% UA in glass-distilled acetone (somewhat altered from [2] and [6]) and infiltrated in Lowicryl HM20.

Imaging was accomplished using the SECOM platform with a 40x/0.95 NA objective, mounted on a Verios 460L SEM (FEI). Fluorescence excitation was completed using a “Pinkel” configuration multiband filter set and LED excitation of 474 and 554 nm for GFP and mCherry, respectively.

A sCMOS camera was used to collect images. SEM imaging was conducted at an acceleration voltage of 3 kV by using the in-column detector (ICD).

Results

Figure 1. Results

References

  1. Peddie, C. J., Blight, K., Wilson, E., Melia, C., Marrison, J., Carzaniga, R., Domart, M.-C., O’Toole, P., Larijani, B., & Collinson, L. M. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy, 143, 3-14, 2014.
  2. Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., & Briggs, J. A. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. The Journal of Cell Biology, 192(1), 111-119, 2011.
  3. Brama, E., Peddie, C. J., Jones, M. L., Domart, M. C., Snetkov, X., Way, M., Larijani, B., & Collinson, L. M. Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope. Journal of Chemical Biology, 2015.
  4. Johnson, E., Seiradake, E., Jones, E. Y., Davis, I., Grünewald, K., & Kaufmann, R. Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Scientific Reports 5, 9583, 2015.
  5. Paez-Segala, M. G., Sun, M. G., Shtengel, G., Viswanathan, S., Baird, M. A., Macklin, J. J., Patel, R., Allen, J. R., Howe, E. S., Piszczek, G., Hess, H. F., Davidson, M. W., Wang, Y., & Looger, L. L. Fixation-resistant photoactivatable fluorescent proteins for CLEM. Nature Methods 12(3), 215-218, 2015.
  6. Avinoam, O., Schorb, M., Beese, C. J., Briggs, J. A., & Kaksonen, M. Endocytic sites mature by continuous bending and remodeling of the clathrin coat. Science, 348 (6241), 1369-1372, 2015.

This information has been sourced, reviewed and adapted from materials provided by Delmic B.V.

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