In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

Abstract

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

The promise and the challenges of cryo‐electron tomography

“…Recently, microfluidics cryofixation devices have been used to address this issue. They are able to arrest a particular cellular state within milliseconds by enabling vitrification on the imaging stage [32].…”

“…High‐pressure freezing devices are limited to ~ 150 µm thickness of material or alternatively up to a few hundred µm with double‐sided cooling devices [22]. With microfluidic freezing platforms, the sample thickness is given by the channel depth of 20 µm [32]. Freezing of bulk specimens has not seen substantial improvements in the past two or three decades and would need revisiting, to make tissue vitrification routine.…”

Challenges and triumphs in cryo-electron tomography

“…The authors were able to target fluorescent lipid droplets in cells grown and plunge-frozen on EM grids ( Figure 5 C) ( Arnold et al, 2016 ). Combining all of these methods is not a simple process, but more streamlined workflows are being developed ( Fuest et al, 2019 ; Wu et al, 2020 ), and it is only a matter of time before it is commonplace. For instance, recently a correlative approach was described that starts with cryo-confocal microscopy, and is followed by using a cryo-mill-and-view method for volume imaging, and finally a lamella is prepped for cryo-ET at a chosen point within the volume ( Wu et al, 2020 ) ( Figures 5 D–5F).…”

“… Graphical abstract

Timeline of major cryo-EM milestones discussed in the text. Citations for each event are as follows: (1) (1979): Heuser et al., 1979 ; (2) (2004): Xuong et al, 2004 ; (3) (2007): Marko et al., 2007 ; (4) (2008): NA; (5) (2012): Brilot et al, 2012 and Campbell et al, 2012 ; (6) (2014/VPP): Danev et al., 2014 ; (7) (2014/cryo-PALM): Chang et al., 2014 ; (8) (2018): Fuest et al., 2018 ; (9) (2019): Fuest et al, 2019 ; (10) (2017): Chen et al, 2017 . …”

In situ Electron Microscopy of Complex Biological and Nanoscale Systems: Challenges and Opportunities

“…This is analogous to the well-developed single-particle image processing algorithms for cryo-EM (Punjani et al, 2017). To study the dynamics of proteins (Zheng et al, 2003;Chen and Lee, 2010;Lutz-Bueno et al, 2016;Knoška et al, 2020) and nanoparticles (Ghazal et al, 2016), microfluidic platforms have been coupled to optical (Zheng et al, 2003;Sakai et al, 2019), fluorescence microscopies (Chen and Lee, 2010;Ding et al, 2016;Hua et al, 2016;Tian et al, 2019), cryo-EM (Fuest et al, 2019;Mäeots et al, 2020), and small-angle Xray scattering (Lutz-Bueno et al, 2016;Anaraki et al, 2020). Some examples include capturing the transient conformations of DNA during self-assembly (Wang et al, 2020), imaging protein crystallization (Zheng et al, 2003) (Figure 6A) or adsorption (Yu et al, 2020), probing intracellular communication via gap junctions (Chen and Lee, 2010) (Figure 6B), mapping the dynamics of amyloid formation (Lutz-Bueno et al, 2016;Saar et al, 2016) (Figure 6C), chemical and molecular mapping of live biofilms (Ding et al, 2016) (Figure 6D), as well as imaging biomineralization and bio-sedimentation (He et al, 2019;Narayanan et al, 2020).…”