Direct correlation of fluorescence microscopy, electron microscopy, and NanoSIMS stable isotope imaging on a single biological tissue section.

Session

LST.5 - Correlative Microscopy across the scales

Authors

Dr Loussert Fonta celine (1), PhD Toullec Gaelle (1), Dr Aby Paraecattil Arun (2), Dr Escrig Stephane (1), Prof Meibom Anders (1), Dr Krueger Thomas (1)

Affiliations

1. EPFL
2. Green Light Solution SA

Keywords

Correlative microscopy

Cryosectioning

Fluorescence microscopy

NanoSIMS

Transmission Electron Microscopy

Abstract text

State-of-the-art studies of complex biological processes often combine multiple experimental methods and employ a variety of optical-, electron-, and ion microscopy technics to image tissues at the single-cell level. These techniques have enabled advances in biology and life sciences by providing information about the localization and distribution of specific molecules (e.g., fluorescence in situ hybridization (FISH)¹ and immunolabelling²), as well as anabolic turnover and cellular exchange processes (e.g., NanoSIMS^3,4), together with ultrastructural information even in the most complex biological tissues. When these techniques are used in combination, the approach is commonly referred to as correlative microscopy⁵. 

Although correlative microscopy now features prominently in studies of biological tissue, important limitations still exist with regard to how well different types of imaging information can be correlated. These limitations are primarily due to sample preparation constraints. For example, in Correlative Light- and Electron Microscopy (CLEM) most protocols involve two main steps, starting with live or fixed-cell fluorescence imaging, followed by sample preparation for electron microscopy (EM)⁶. Classical EM sample preparation involves chemical fixation, heavy metal staining, dehydration with solvents, resin embedding, and subsequent (thin-)sectioning. Following this treatment, the soluble compounds in the cell (i.e., cytosolic components) are either thoroughly displayed or completely lost and the tissue has shrunk substantially; altogether it is a very different sample compared to the material that was observed previously by fluorescence microscopy. To overcome this problem, a sample preparation method developed by Prof. Tokuyasu⁷ is often used in CLEM^8,9, permitting fluorescence and electron microscopy to be carried out on the same section⁸. This method involves chemical fixation similar to classical preparation, cryo-sectioning, and thawing at room temperature. By avoiding dehydration and resin embedding, the Tokuyasu method minimizes morphological artifacts (in particular tissue shrinkage) and chemical modifications at the molecular level, preserving the (auto-)fluorescence properties of the sample⁹ and (to variable degree) its antigenicity, thus permitting a large range of antibody-labeling.

In the last 10-15 years, ultrahigh resolution (ca. 100 nm lateral resolution) quantitative ion microprobe imaging (NanoSIMS), combined with experiments that introduce stable isotopic (e.g., ¹³C and ¹⁵N) and/or elemental labels into a tissue, has made it possible to study anabolic turnover and track specific molecules with sub-cellular resolution. NanoSIMS imaging has found applications across numerous disciplines within the environmental, biological, and life sciences^3,4,10. Nevertheless, it is still not possible to correlate information obtained with all three imaging technics (i.e., fluorescence microscopy, electron microscopy, and NanoSIMS) from one-and-the-same section of a biological tissue. Such a capability would represent a major breakthrough in correlative microscopy¹¹ because it would permit structural, molecular, and anabolic/metabolic information to be directly correlated at the (sub-)cellular level. Here we present a methodology, building upon the Tokuyasu¹² method, that enables direct correlation of (immuno)fluorescent microscopy, (immuno)TEM, and NanoSIMS ultra high-resolution stable isotopic mapping of the same biological tissue section. 

We demonstrate this new level of correlative microscopy on tissue from a symbiotic coral (here Stylophora pistillata). Reef building corals are highly complex organisms that consist of a various range of tissue- and cell types. In these organisms, the ectoderm and endoderm of both oral and aboral layers are separated by a hydrogel-like matrix. Many of the endodermal cells host symbiont photosynthesizing dinoflagellate algae inside symbiosomes¹³. A diverse community of bacteria¹⁴ adds to the complexity of this symbiotic organism, which is referred to as the “coral holobiont”. The structural complexity of symbiotic corals thus represents a methodological challenge. At the same time, it conveniently provides the opportunity to demonstrate our new sample preparation- and correlative microscopy approach on a range of different matrices within a single biological tissue.

 

       

References

1.   Huber, D., Voith von Voithenberg, L. & Kaigala, G. V. Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH? Micro and Nano Engineering 1, 15–24 (2018).

2.   de Matos, L. L., Trufelli, D. C., de Matos, M. G. L. & da Silva Pinhal, M. A. Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice. Biomark Insights 5, 9–20 (2010).

3.   Hoppe, P., Cohen, S. & Meibom, A. NanoSIMS: Technical Aspects and Applications in Cosmochemistry and Biological Geochemistry. Geostand Geoanal Res 37, 111–154 (2013).

4.   Nuñez, J., Renslow, R., Cliff, J. B. & Anderton, C. R. NanoSIMS for biological applications: Current practices and analyses. Biointerphases 13, 03B301 (2018).

5.   Ando, T. et al. The 2018 correlative microscopy techniques roadmap. Journal of Physics D: Applied Physics 51, 443001 (2018).

6.   Hauser, M. et al. Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities. Chemical Reviews 117, 7428–7456 (2017).

7.   Tokuyasu, K. T. Immunochemistry on ultrathin frozen sections. The Histochemical Journal 12, 381–403 (1980).

8.   Oorschot, V. M. J., Sztal, T. E., Bryson-Richardson, R. J. & Ramm, G. Immuno Correlative Light and Electron Microscopy on Tokuyasu Cryosections. in Methods in Cell Biology vol. 124 241–258 (Elsevier, 2014).

9.   Loussert Fonta, C. et al. Analysis of acute brain slices by electron microscopy: A correlative light–electron microscopy workflow based on Tokuyasu cryo-sectioning. Journal of Structural Biology 189, 53–61 (2015).

10. Gyngard, F. & Steinhauser, M. L. Biological explorations with nanoscale secondary ion mass spectrometry. J. Anal. At. Spectrom. 34, 1534–1545 (2019).

11. Decelle, J. et al. Subcellular Chemical Imaging: New Avenues in Cell Biology. Trends in Cell Biology S0962892419302211 (2020) doi:10.1016/j.tcb.2019.12.007.

12. Tokuyasu, K. T. A technique for ultracryotomy of cell suspensions and tissues. The Journal of Cell Biology 57, 551–565 (1973).

13. Wakefield, T. S. & Kempf, S. C. Development of Host- and Symbiont-Specific Monoclonal Antibodies and Confirmation of the Origin of the Symbiosome Membrane in a Cnidarian–Dinoflagellate Symbiosis. The Biological Bulletin 200, 127–143 (2001).

14. Rosenberg, E., Koren, O., Reshef, L., Efrony, R. & Zilber-Rosenberg, I. The role of microorganisms in coral health, disease and evolution. Nat Rev Microbiol 5, 355–362 (2007).