The Importance of Sample Processing for Correlative Imaging (or, Rubbish In, Rubbish Out)

Peddie, Christopher J.; Schieber, Nicole L.

doi:10.1002/9781119086420.ch3

Cited by 4 publications

(12 citation statements)

References 165 publications

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“…Throughout the complete workflow, care needs to be taken during the sample preparation regarding distortions of the tissue 19 . Due to the abundance of blood vessels in liver tissue, the sections are prone to distortions such as those caused by the pressure of a coverslip (LM) and extensive processing and dehydration (EM).…”

Section: Discussionmentioning

confidence: 99%

“…For CLEM in three dimensions, a workflow that allows the tracking of a region of interest (ROI) from LM to EM is most imperative, as even small alterations can cause a loss of navigation throughout the ultrastructure due to the gap in resolution between the methods. Reflected in this, sample preparation is vital as it needs to be fine‐tuned, not only to assure the best possible results in the two imaging modalities, but also to incorporate a strategy to relocate an ROI imaged in LM in the EM 18–20 . The strategy presented here depends on fiducial markers on two levels: markers pointing to the ROI, enabling localisation of the ROI imaged in LM under the electron microscope after the intermittent processing steps, and markers that are visible in both LM and EM that can be used as landmarks to generate overlays of the data correlating fluorescent labels to the underlying ultrastructure.…”

Section: Introductionmentioning

confidence: 99%

“…The strategy presented here depends on fiducial markers on two levels: markers pointing to the ROI, enabling localisation of the ROI imaged in LM under the electron microscope after the intermittent processing steps, and markers that are visible in both LM and EM that can be used as landmarks to generate overlays of the data correlating fluorescent labels to the underlying ultrastructure. These fiducials have to be precise, independent of the protein or structure of interest and ideally be present throughout the 3D data without altering the native state of the tissue 19,21,22 …”

Section: Introductionmentioning

confidence: 99%

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A workflow for 3D‐CLEM investigating liver tissue

Kremer

Hamme

Bonnardel

et al. 2020

Journal of Microscopy

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Summary Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume‐EM techniques such as serial blockface scanning electron microscopy (SBF‐SEM) and Focussed Ion Beam‐SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume‐EM with high‐resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high‐resolution confocal microscopy with SBF‐SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D‐CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three‐dimensional correlative studies. Lay Description As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10−9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high‐resolution contextual information provided by the electron m...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A workflow for 3D‐CLEM investigating liver tissue

Kremer

Hamme

Bonnardel

et al. 2020

Journal of Microscopy

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“…Its power relies on its ability to bring together data from a variety of cell features captured using different contrasting methods. This way, high lateral, axial and spatiotemporal resolutions can be achieved when imaging the same cellular areas and capturing structures or processes across different scales at different time points and over a range of wavelengths, while also delivering volumetric information [1][2][3][4] . Since correlative imaging was first reported in 1965 5 , when the term correlative light and electron microscopy (CLEM) was coined, techniques have evolved and matured but have also led to challenges in the quest to capture and cross-reference native cell structures using different microscopy platforms [6][7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

“…These data may well originate from the exact same area within a cell; however, they are likely to bear no similarity in the recorded images, leaving the researcher without reference points to allow the useful association of the information captured. This brings about the absolute need for positional markers (fiducials) visible across the correlative scheme that can serve as universal points of reference for 3D imaging data alignment 1,13,[36][37][38][39] .…”

Section: Introductionmentioning

confidence: 99%

Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures

et al. 2021

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This protocol describes sample preparation strategies for correlative 3D cryo-SIM and cryo soft X-ray tomography. In addition, the authors provide a direct comparison and recommendations regarding the selection and use of fiducials for 3D correlation. TWEET A new protocol for sample preparation and fiducial selection for correlative cryo-SIM and cryo-soft X-ray tomography. #CLXT #cryoimaging #cellstructure #correlativeimaging COVER TEASER Correlative cryo-SIM and cryo-soft X-ray tomographyAbstract 3D correlative microscopy methods have revolutionised biomedical research allowing the acquisition of multi-dimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometre resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample resulting in the immediate requirement for protocols that facilitate hassle-free unambiguous cross correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialisation regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 hours and 3.5 days to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

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Volume electron microscopy

Peddie

Genoud

Kreshuk

et al. 2022

Nat Rev Methods Primers

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Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.

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The Importance of Sample Processing for Correlative Imaging (or, Rubbish In, Rubbish Out)

Cited by 4 publications

References 165 publications

A workflow for 3D‐CLEM investigating liver tissue

A workflow for 3D‐CLEM investigating liver tissue

Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures

Volume electron microscopy

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