Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation

Abstract

Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

“…To be noted, besides the non-integrated cryo-CLEM systems, another cryo-CLEM concept has been recently developed, which integrates the fluorescence imaging system into the cryo-FIB/cryo-SEM chamber. These integrated systems include PIE-Scope 22 , iFLM 52 , METEOR 53 , and the coincident three-beam microscope 54 , as well as the very recently published ELI-TriScope 55 and CLIEM 56 by us and our colleagues. Besides avoiding specimen transfer during microscopies to minimize the risks of specimen damage and ice contamination, these systems can also achieve correlative imaging without the need of fluorescent fiducial markers after a proper pre-calibration 54 – 56 , therefore significantly increasing the cryo-CLEM workflow efficiency.…”

“…These integrated systems include PIE-Scope 22 , iFLM 52 , METEOR 53 , and the coincident three-beam microscope 54 , as well as the very recently published ELI-TriScope 55 and CLIEM 56 by us and our colleagues. Besides avoiding specimen transfer during microscopies to minimize the risks of specimen damage and ice contamination, these systems can also achieve correlative imaging without the need of fluorescent fiducial markers after a proper pre-calibration 54 – 56 , therefore significantly increasing the cryo-CLEM workflow efficiency. Our ELI-TriScope 55 provides another unique advantage to monitor the fluorescence of the target during cryo-FIB milling, therefore minimizing the off-target effect induced by cryo-FIB milling and accompanied with the cryo-lamella deformation.…”

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

“…The recent fusion of fluorescence microscopy and FIB-SEM techniques heralds a significant advancement. This integration not only minimizes sample contamination but also bolsters the practicality of cryo-ET methodologies. , …”

“…This integration not only minimizes sample contamination but also bolsters the practicality of cryo-ET methodologies. 370,371 Once cryo-lamellas are prepared, they are transferred to the TEM to obtain tomographic images. The sample is tilted to acquire a series of projection images, which are then computationally combined to generate a 3D tomographic volume.…”

Depth-dependent scaling of axial distances in light microscopy

“…The use of air objectives is a straight-forward choice, as the specimen resides in a vacuum chamber for electron microscopy. In cryogenic fluorescence microscopy the specimen is cooled to temperatures below 120 K, which makes the use of a non-touching air objectives favorable, as any other (touching) immersion objectives are challenging in terms of engineering [9][10][11][12].…”