CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging

Michael A Phillips,Richard M Parton,David I Stuart,Ana Palanca,Ilan Davis,Martin J Booth,Manuel Garcia-Moreno,Ian M Dobbie,David Miguel Susano Pinto,John W Sedat,Alfredo Castello,Maria Harkiolaki,Ilias Kounatidis

doi:10.1364/optica.393203

Abstract

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.

Highlights

Imaging methods for cells and tissues have progressed rapidly in the past decade, providing unrivalled opportunities for new insights into biological molecular mechanisms [1,2,3]
The ultrastructural information becomes informative when the distributions of specific molecules are added. This can be done by imaging the same sample in fluorescence and precisely painting the fluorescence signal onto the electron microscopy (EM) or X-ray tomography deduced morphology in the so-called correlative methods, producing correlative light and electron microscopy (CLEM) or correlative light and X-ray tomography [6]
Molecular localization in EM has been possible for many years [7], often through difficult techniques, for example, the use of antibodies coupled to different sized gold particles in Correlative fluorescence and ultrastructural microscopy have successfully been applied to the localization of molecules in plastic sections [8,9,10]

Summary

Introduction

Imaging methods for cells and tissues have progressed rapidly in the past decade, providing unrivalled opportunities for new insights into biological molecular mechanisms [1,2,3]. Molecular localization in EM has been possible for many years [7], often through difficult techniques, for example, the use of antibodies coupled to different sized gold particles in Correlative fluorescence and ultrastructural microscopy have successfully been applied to the localization of molecules in plastic sections [8,9,10]. Using such sections, it has even been possible to carry out super-resolution fluorescence imaging using singlemolecule localization microscopy (SMLM) techniques [11,12,13]. To date such approaches have all involved chemical fixation, which is subject to potential artefacts

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Discussion

Conclusion