Cryofixation during live‐imaging enables millisecond time‐correlated light and electron microscopy

Fuest, Marie; Nocera, Giovanni Marco; Modena, Mario M.; Riedel, Dietmar; Mejia, Yara X.; Burg, Thomas P.

doi:10.1111/jmi.12747

Cited by 17 publications

(27 citation statements)

References 49 publications

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“…In chemical fixation protocols, the specimen is usually immobilized by exposure to a fixative during imaging, giving a time correlation close to 0s (Stepanek & Pigino, 2017), but at the same time exposes the sample to the risk of introducing artifacts. An interesting alternative, that is yet to be commercialized is a microfluidic chamber that allows cryoarrest during imaging (FUEST et al, 2018). Currently, no automated solution exists for CLEM of thick samples.…”

Section: Cryo-capturing Fast and Small Biological Eventmentioning

confidence: 99%

HPM Live μ for a full CLEM workflow

Heiligenstein¹,

Beer

Heiligenstein³

et al. 2020

Preprint

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With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To explore further the potential of cryo-fixation, we developed a high pressure freezing (HPF) system that allows vitrification under different environmental parameters and apply it in different CLEM workflows. In this chapter we introduce our novel HPF live μ instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

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Section: Cryo-capturing Fast and Small Biological Eventmentioning

confidence: 99%

HPM Live μ for a full CLEM workflow

Heiligenstein¹,

Beer

Heiligenstein³

et al. 2020

Preprint

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“…In a broad sense, correlative microscopy refers to any combination of microscopy techniques (Karreman et al, 2016), microscopic X-ray computed tomography of excised tissue (Karreman et al, 2017), transmission electron microscopy (TEM) (Fuest et al, 2018), atmospheric or environmental scanning electron microscopy (SEM) (Peckys and de Jonge, 2015;Sato et al, 2019), cathodoluminescence of nanoparticles in SEM (Glenn et al, 2012), cryo-electron tomography (Tao et al, 2018), array tomography of electron microscopy (Burel et al, 2018;Collman et al, 2015;Micheva and Smith, 2007), X-ray holography, X-ray scanning diffraction (Bernhardt et al, 2018), X-ray fluorescence imaging and mass spectrometry imaging (Decelle et al, 2020). Some authors emphasized this integration of information from multiple imaging methods, by proposing a "morphomics" notation (Lucocq et al, 2015).…”

Section: Correlative Microscopymentioning

confidence: 99%

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

Kawano

Kakazu

Iwabuchi

et al. 2020

Preprint

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ABSTRACTBackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems.New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets.ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level.Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems.ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.

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“…We have recently shown a microfluidic based technology that is able to cryofix samples directly in the light microscope 22 . This technology enables millisecond time correlation between live imaging and electron microscopy, where the time correlation is limited only by the sample freezing time.…”

Section: Introductionmentioning

confidence: 99%

“…To date, microfluidic cryofixation has been limited to room temperature electron microscopy studies following freeze substitution and resin embedding of cryofixed samples 22 . Accessing the native in situ cellular structures with high resolution cryo-ET was a substantial challenge due to the inability to form electron transparent cryo-sections from samples embedded in the microchannels.…”

Section: Introductionmentioning

confidence: 99%

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

Fuest

Schaffer

Nocera

et al. 2019

Sci Rep

Self Cite

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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.

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Cryofixation during live‐imaging enables millisecond time‐correlated light and electron microscopy

Cited by 17 publications

References 49 publications

HPM Live μ for a full CLEM workflow

HPM Live μ for a full CLEM workflow

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

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

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