S E C O M

application note

Cellular Biology:
In-Resin Fluorescence Using GFP
and mCherry

S E C O M

application note

Cellular Biology:
In-resin Fluorescence Using GFP
and mCherry
Correlative light and electron microscopy (CLEM) is a powerful technique forstudying cellular processes on an ultrastructural level. The SECOM platform,a fluorescence microscope designed to be integrated with a scanning electron microscope, can simplify experiments involving CLEM by providing afully automated overlay procedure and seamless switching between fluorescence and electron microscopy.
Sample preparation, however, remains the crux of any experiment. To
achieve the best overlay accuracy, one ideally acquires both the fluorescence and electron images on the same resin -embedded section [1,2]. This
does pose a challenge with regards to sample preparation. Fortunately,
recent advances in sample preparation have led to a range of protocols that
demonstrate in-resin fluorescence of fluorescent proteins [1 -5].

Two studies report that imaging GFP and YFP in vacuum has a large influence on the fluorescent intensity. From [3]: “ Firs t , w e o bs e rve d a d ro p influorescence intensity as the vacuum pressure decreases due to extraction ofwater from the sample, and which can be reversed by re -introducing water into
the system at partial vacuum pressure or atmospheric pressure. Second, weshow that although fluorescence intensity is reduced at a partial pressure of 200
Pa (created using water vapour), the FP intensity is remarkably stable andresistant to photobleaching during imaging. Finally, we show that holding IRF
sections in vacuum leads to very minor losses in fluorescence over time. ”

DELMIC BV | +31 (0)15 744 0158| info@delmic.com | www.delmic.com

S E C O M

Here we used Hela Kyoto cells stably expressing GalNAC -T2-GFP andHistone 2B-mcherry. The cells were grown on carbon coated sapphiredisks and high pressure frozen. The cells were then freeze substituted
with 0.1% UA in glass distilled acetone (slightly modified from [2] and[6]) and infiltrated in Lowicryl HM20.
Imaging was performed using the SECOM platform with a 40x/0.95 NA
objective, mounted on a Verios 460L SEM (FEI). Fluorescence excitation
was performed using a ‘Pinkel’ configuration multiband filter set and LEDexcitation of 474 nm and 554 nm for GFP and mCherry respectively.
Images were collected using an sCMOS camera. SEM imaging was performed with a 3 kV acceleration voltage and using the in -column detector (ICD).

Figure 1 Results

References
1.
2.
3.
4.
5.
6.

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 andelectron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy, 143, 3-14, 2014.
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.
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.
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.
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.
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.

DELMIC BV | +31 (0)15 744 0158| info@delmic.com | www.delmic.com

DELMIC B.V. is a company based in Delft, the Netherlands that
produces correlative light and electron microscopy solutions.
DELMIC's systems cater to a broad range of researchers infields ranging from nanophotonics to cell biology.
The SECOM platform is a fluorescence microscope made to be
integrated with a scanning electron microscope produced byDELMIC, that enables extremely fast correlative microscopy,
with the highest optical quality and overlay accuracy.
For questions regarding this note, contact our SECOM Application
Specialist at: voortman@delmic.com
For more SECOM application notes, see:delmic.com/secom/application_notes.php
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info@delmic.com