In CLEM experiments biological samples are typically first investigated by light microscopy (LM) to locate a region of interest. Next, nanometer-scale imaging can be achieved by electron microscopy (EM). This results in identification of biomolecules within the ultrastructure and/or the possibility to correlate ultrastructure with preliminary live-cell dynamics on biomolecules [1].

We have developed an integrated CLEM with which widefield fluorescence microscopy (WF-FM) and EM can be performed in principle simultaneously [2]. Integration of these two modalities allows for fast ROI retrieval [3], optional fast switching between modalities increasing CLEM throughput, high (5 nm) correlation accuracy for the overlay [4], and novel imaging applications [5]. However, for WF-FM the depth correlation for thick specimen or live cells contained in a dedicated liquids sample holder [6] is relatively poor. WF-FM images through the entire sample and SEM only the upper ~100 nm (depending on e-beam energy). We therefore integrated a Confocal Laser Scanning Microscope (CLSM) into a scanning electron microscope (SEM) via a Delmic Secom stage (Fig. 1-4). This system will allow imaging with improved depth-correlation by suppression of out-of-focus light contributions and will provide 3D optical information in CLEM.

We present our integrated CLSM SEM, using a commercial confocal scanner unit. We also demonstrate the procedure for alignment of the CLSM and SEM axes, needed to image the same area with both modalities. Lastly, the results of proof-of-principle experiments are presented, showing optical sectioning in thick specimen such as resin blocks or whole cells for improved LM-SEM correlation along the axial direction.    

[1] P. de Boer, J.P. Hoogeboom, B.N.G. Giepmans, Correlated light and electron microscopy: ultrastructure lights up!, Nature methods, 12, 503-513 (2015)

[2] A.C. Zonnevylle, R.F.C. Van Tol, N. Liv, A.C. Narvaez, A.P.J. Effting, P. Kruit, J.P. Hoogenboom, Integration of a high-NA light microscope in a scanning electron microscope, Journal of Microscopy, 252-1, 58-70 (2013)

[3] N. Liv, A.C. Zonnevylle, A.C. Narvaez, A.P.J. Effting, P.W. Voorneveld, M.S. Lucas, J.C. Hardwick, R.A. Wepf, P. Kruit, J.P. Hoogenboom, Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy, PLOS one, 8-2, e55707 (2013)

[4] M.T. Haring, N. Liv, A.C. Zonnevylle, A.C. Narvaez, L.M. Voortman, P.Kruit, J.P.Hoogenboom (submitted)

[5] J. Sueters, N. Liv, J.P. Hoogenboom, Using Advanced Correlative Microscopy to Study Complex Biological Samples, Encyclopedia of analytical chemistry, a9473, (2016, in press).

[6] N. Liv, D.S.B. Van Oosten Slingeland, J-P. Baudoin, P. Kruit, D.W. Piston, J.P. Hoogenboom, Electron microscopy of living cells during in situ fluorescence microscopy, ACS Nano, 10-1, 265-273 (2016)   

Figures:

Integrated CLEM setup with FEI Verios SEM and Delmic secom optical stage

Vacuum parts of the CLEM setup

Optical components of CLEM setup

Schematic representation of the CLEM setup

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EMC Abstracts - https://emc-proceedings.com/abstract/improving-3d-correlation-in-integrated-correlated-light-and-electron-microscopy-using-confocal-laser-scanning-microscopy/