微米到奈米的關聯應用 - 研討會 非破壞3D X-ray 顯微鏡與生物電子顯微鏡聯用發展

In T.B.  Willingham et al. 2020, Dr. Glancy's group uses volume electron microscopy with FIB-SEM to unveil that striated muscle cells form a continuous myofibrillar matrix with frequent, branching sarcomeres. Their work examines changes in branching during postnatal development and in different muscle types. They suggest a new theory for how force is generated based on a mesh-like myofibrillar network rather than many individual, parallel myofibrils.

Continuing their work from T.B. Willingham et al. 2020 mentioned above, Dr. Glancy and team looked into the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching. In P.T. Ajayi et al. 2022,  they present 3D electron microscopy evidence which indicates fruit flies have a myofibrillar connectivity on/off switch that is regulated by both cell-type dependent and independent mechanisms.

Specimen charging, particularly in samples containing large regions of bare resin, results in a significant degradation in image quality and distortion. Typically, charging is mitigated by applying variable pressure, however this is at the expense of signal-to-noise ratio and resolution.

ZEISS Focal Charge Compensation eliminates specimen charging. A gas injection system is precisely located above the sample. Nitrogen is guided directly onto the block-face surface while the chamber is maintained under high vacuum. This eliminates charging and assures high image quality. The needle retracts automatically during the cutting cycle, so the workflow is uninterrupted and high acquisition rates are maintained.

ZEISS software combines the individual Volutome hardware components to make the serial block-face workflow smooth and easy-to-use. The cutting operation as well as the imaging process are controlled by ZEISS ZEN core. ZEN core workbenches provide intuitive structure to control setup, sample to knife approach and parameters for cutting and image acquisition.

Once the data is collected and the pre-calculation is applied for stitching and z-stack alignment, the results can be visualized and processed with ZEISS arivis Pro.

Take your results a step further, with software from the ZEISS arivis product family you can an-notate, segment, and analyze your data – getting the most information out of your images.  

High resolution imaging is necessary to visualize the ultrastructure of cells and cellular components. Samples with large areas of bare resin are particularly prone to charging. Focal Charge Compensation avoids the charging effects and delivers high-quality images. The sensitivity of ZEISS Volume BSD permits low kV imaging without sacrificing image contrast or acquisition time. Under these conditions, various cellular components, such as mitochondria, Golgi, and even vesicles can be identified and analyzed.

Genetically modified stem cells

• Pixel size: 10 nm
• Cutting thickness: 30 nm
• Dimensions: 51 µm × 51 µm x 15 µm (~550 sections)
• EHT: 1.5 kV / Ip: 100 pA
• Dwell time: 2.8 µs
• Acquired with ZEISS GeminiSEM 460

Volume electron microscopy enables imaging of much larger sample sizes, making visualization of larger tissue sections a more routine application for life scientists across many disciplines. Whether you work with tumors and biopsies, organ or tissue sections, organoids, embryos of model organisms and more, serial block-face imaging allows large sample volumes to be imaged and analyzed within a broader 3D context. Investigate your samples in healthy or diseased states, or examine the effects of metabolic changes, genetic factors, drug treatments, and more.

Mouse skeletal muscle

• Pixel size: 3 nm
• Cutting thickness: 100 nm
• Dimensions: 18 μm × 15 μm × 25 μm (250 sections)
• EHT: 2 kV / Aperture: 20 μm, high current
• Dwell time: 1 µs
• Acquired with ZEISS GeminiSEM 360

With more X-ray photons available on ZEISS Xradia 600-series Versa, you can now achieve even faster time to results for varied samples without compromising resolution. Unique to ZEISS Xradia 630 Versa is the 40X-Prime (40X-P) objective lens.

ZEISS Xradia 630 Versa XRM, with the higher energy capabilities of the exclusive 40X-Prime (40X-P) objective, enables you to push the limits of submicron imaging like never before. Known for their ability to achieve resolution at a Distance (RaaD™), ZEISS Xradia Versa platforms allow high resolution imaging of a wide array of sample types and sizes over a long range of length scales.

With 40X-P, the system achieves unparalleled resolution performance of 450-500 nm across the full range of source voltage, from 30 kV to 160 kV, defining RaaD 2.0. Unlocking entirely new application capabilities for researchers, the ZEISS 40X-P objective enables ZEISS Xradia 630 Versa to push industry standards of submicron imaging resolution.

The flat panel extension (FPX), standard on your ZEISS Xradia 630 Versa X-ray microscope, further increases the versatility of the system, directly supporting the Advanced Reconstruction Toolbox’s AI-based DeepScout for deep learning and neural network training. Leverage FPX to perform a low resolution, large field of view, "scout” scans, and identify interior regions for higher resolution “zoom” scans on a variety of different sample types. The Volume Scout workflow streamlines this process within NavX.

MARS is a reconstruction algorithm that is aware of the constituents within a reconstruction. A challenge in X-ray reconstruction in a lab setting is that imaging with a polychromatic source creates different X-ray energies to generate a phenomenon called beam hardening. This effect is particularly challenging when your material is very dense and embedded in relatively less dense material. MARS tells the reconstruction system how to compensate for the effect of extreme beam hardening in the regions between very dense objects. This is important in applications like biomaterials, where you might be looking at implants next to bone or tissue. Or electronics where extremely dense solder balls appear next to other less dense materials on a printed circuit board, generating strong artifacts. MARS reconstructs your images to compensate for these effects. ​

Biomedical metal implant in bone. Without MARS, left. With MARS, right.​