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.
Highlights
By contrast, cryo-EM images frozen, hydrated samples in a near native state below the glass transition temperature of water
We demonstrate cryofixation during live imaging followed by cryo-transmission electron tomography
The roundworm C. elegans was used as a model system
Summary
Cryo-EM images frozen, hydrated samples in a near native state below the glass transition temperature of water. Innovative studies have worked to bypass this limitation by initiating a desired reaction with a light or electrical stimulus and carefully timing or programming cryofixation to occur a few to tens of milliseconds after the external trigger[10,17,18,19,20,21] These methods, do not yet allow uninterrupted live imaging of the dynamic process up to cryofixation. This technology enables millisecond time correlation between live imaging and electron microscopy, where the time correlation is limited only by the sample freezing time This technique is of interest for studying rare, highly dynamic, or directional processes by real time selection and arrest of a desired biological state. The combination of cryo-FIB sample preparation with advanced transmission electron microscopy (TEM) methods[29,30,31,32,33] has recently enabled imaging and 3D reconstruction of macromolecular complexes in their native cellular context with molecular resolution[34,35]
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